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WO2025186779A1 - Oncolytic viruses expressing immunomodulators and use for treating advanced solid tumor - Google Patents

Oncolytic viruses expressing immunomodulators and use for treating advanced solid tumor

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Publication number
WO2025186779A1
WO2025186779A1 PCT/IB2025/052481 IB2025052481W WO2025186779A1 WO 2025186779 A1 WO2025186779 A1 WO 2025186779A1 IB 2025052481 W IB2025052481 W IB 2025052481W WO 2025186779 A1 WO2025186779 A1 WO 2025186779A1
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WIPO (PCT)
Prior art keywords
hsv
individual
virus
amino acid
acid sequence
Prior art date
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Pending
Application number
PCT/IB2025/052481
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French (fr)
Other versions
WO2025186779A8 (en
Inventor
Katherine Anne THORNTON
Aisha N. Hasan
Josh LAURING
James Spivey
Julia Billiard
James G. Greger, Jr.
Douglas STEINBACH
Parul Gupta
Jason ALIGO
Roland Knoblauch
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Janssen Biotech Inc
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Janssen Biotech Inc
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Publication of WO2025186779A1 publication Critical patent/WO2025186779A1/en
Publication of WO2025186779A8 publication Critical patent/WO2025186779A8/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/763—Herpes virus
    • 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
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5434—IL-12
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], 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 [IGs], 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • 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
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16632—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent

Definitions

  • the present disclosure relates to methods of treating cancer comprising oncolytic viruses derived from herpes simplex virus 1 (HSV-1), administered in combination with a PD-1 antibody.
  • HSV-1 herpes simplex virus 1
  • Oncolytic virus therapy is a form of immunotherapy that exploits the cytotoxic and/or vector ability of viruses to selectively target and destroy tumor cells. Oncolytic virus therapy can also work to stimulate immune responses against a target tumor or tumors distal to a target tumor. Safety issues limited the use of live, infectious viruses in cancer patients, but the development of robust genetic engineering has allowed the field to mature by the development of improved viruses (Kelly and Russell, Mol. Ther. 2007 Apr; 15(4): 651-9). Oncolytic viruses may be engineered to have enhanced selectively for tumor cells (for example, by enhanced cytotoxicity in cancer cells and/or reduced cytotoxicity in normal cells) and to express therapeutic payloads, such as immunostimulatory proteins.
  • HSV Herpes simplex viruses
  • HSV has a broad host cell range in humans, a short replication cycle, a large genome which is amenable to multiple payload genes, and effective antiviral options to control infection (Sanchala et al., Front. Pharmacol. 2017;8:270).
  • Next generation oncolytic viruses, including those derived from HSV, that exhibit increased safety and increased efficacy are therefore needed in the art. Additionally, whether oncolytic viruses could provide efficacious cancer therapy in combination with other immunotherapy based approaches is poorly understood. Provided herein are methods and compositions that address such and other needs.
  • the oncolytic viruses provided herein trigger an abscopal response to a distant tumor and/or cause an immunological memory of a tumor.
  • FIGS.1A-1B are diagrams of the genome structure and transgene cassettes of the mouse surrogate virus, mJP-OV-2 and the human virus JP-OV-2.
  • the mouse surrogate virus mJP-OV-2 was engineered to express the anti-CTLA-4 antagonist (m ⁇ CTLA-4), CD40 agonist (mCD40ag), and IL- 12 (mscIL-12) payloads from the US 10- 12 locus, and hFLT3L and UL49.5 from the ⁇ 34.5 locus.
  • the virus expresses codon-optimized US11 (hCoUS11) using the US 12 immediate early promoter and endogenous US 11 using late US 11 promoter.
  • hCoUS11 codon-optimized US11
  • the human virus JP-OV-2 was engineered to express the anti-CTLA-4 antagonist (h ⁇ CTLA-4), CD40 agonist (hCD40ag), and IL- 12 (hscIL-12) payloads from the US 10- 12 locus, and hFLT3L and UL49.5 from the ⁇ 34.5 locus.
  • the virus expresses codon-optimized US 11 (hCoUS 11) using the US 12 immediate early promoter and endogenous US 11 using late US 11 promoter. “S” shown in the hexagon represents a stop codon between hCoUS 11 and US 12, which inhibits US12 expression. US12 in grey indicates that the gene does not express.
  • IRL internal repeat long
  • IRS internal repeat short
  • TRL terminal repeat long
  • TRS terminal repeat short
  • UL unique long
  • US unique short.
  • FIGS. 2A-2C show the effect of treatment with an oncolytic HSV-1 virus in combination with a PD-1 inhibitor in a mouse tumor model system.
  • FIG. 2B shows the tumor volume for the treated and untreated tumors and graphed as the mean ⁇ SEM.
  • FIG. 2C shows survival of the treatment groups compared with that of the vehicle control group for oncolytic HSV-1 virus treatment in combination with an anti-PD-1 antibody.
  • IT intratumoral; SEM, standard error of the mean.
  • FIGS. 3A-3C illustrate the design of a Phase 1 study.
  • FIG. 3 A shows a schematic overview of the study including the dose escalation analysis in Part 1 (right) and the dose expansion analysis in part 2 (left).
  • FIG. 3B shows the administration schedule for Part 1.
  • FIG. 3C shows the administration schedule for Part 2.
  • a Refer to Example 2 for full treatment duration description.
  • Additional treatments, up to 8 more doses, may be given with sponsor approval on a case by case basis.
  • Doses may be skipped based on clinical feasibility and medical condition of the patient.
  • FIG. 4 presents a swim lane plot that illustrates the duration of treatment, indicated by the length of the horizontal bars, alongside the best clinical response for individual patients at each dose level, represented by white circles.
  • the response is evaluated according to oncology standards, specifically using RECIST v1.1 criteria, which classifies a response as a reduction in total measurable tumor burden of at least 30%. Additionally, clinical benefit is defined as the presence of stable disease according to RECIST for a minimum duration of 12 weeks. The bars without any white circles have not had a disease evaluation.
  • FIG. 5 A presents a waterfall plot that displays the post- treatment change in total measurable tumor burden according to standard oncology guidelines (RECIST v1.1).
  • the vertical bars represent individual patients and illustrate a dose-dependent effect of JNJ916 on tumor growth control, with effects increasing from left to right across the plot.
  • the first third of FIG. 5 A depicts the 10 6 dose, the second third shows the 10 7 dose, and the final third represents the 10 8 dose.
  • FIG. 5B highlights tumor shrinkage in uninjected lesions (represented by light bars).
  • FIG. 6A illustrates the quantities of each payload protein detected in tumor tissues collected from patient samples, as assessed using Meso Scale Discovery (MSD) assay.
  • MSD Meso Scale Discovery
  • FIG. 6B illustrates the clinical payload expressions in preclinical mouse models.
  • FIG. 6C shows the correlation between the tumoral payload levels and interferon-y.
  • FIGs. 7A, 7B, and 7C display the data for a responding patient at the highest dose level.
  • the drawings presented include CT scans of a representative injected lesion and an uninjected lesion (FIG. 7A), quantification of serum levels of JP-OV-2-encoded immune payloads (FIG. 7B), and the corresponding effects on immune activation, illustrated by serum interferon- ⁇ levels (FIG. 7C).
  • FIG. 8. displays serum levels of interferon-y at various time points during treatment for patients treated at each JP-OV-2 dose level.
  • FIG. 9 depicts the immune microenvironment in paired tumor tissue samples collected from the same lesion. One sample was obtained before the injection of JP-OV-2, and the other was collected after the administration of three JP-OV-2 injections.
  • the present disclosure provides methods of treating solid tumors in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus and a PD-1 antibody to the individual.
  • the oncolytic virus disclosed herein is armed with four immunomodulatory pay loads: human fms like tyrosine kinase 3 ligand (hFLT3L), human cluster of differentiation (hCD)40 agonist (ag), antibody to human cytotoxic T lymphocyte-associated protein 4 ( ⁇ CTLA4), and human single-chain (hsc) interleukin (IL) 12.
  • hFLT3L human cluster of differentiation
  • ⁇ CTLA4 human cytotoxic T lymphocyte-associated protein 4
  • IL interleukin
  • Reference to “about” a value or parameter herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) aspects that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.
  • antibody herein is used in the broadest sense and encompasses various antibody structures (immunoglobulin molecules, fragments of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen under typical physiological conditions), including but not limited to monoclonal antibodies, 4-chain antibodies (such as IgG antibodies), heavy chain antibodies, and antibody fragments thereof so long as they exhibit the desired antigen-binding activity.
  • 4-chain antibodies such as IgG antibodies
  • heavy chain antibodies such as antibodies
  • antibody fragments thereof so long as they exhibit the desired antigen-binding activity.
  • 4-chain antibody is used herein to refer to an antibody or antigen-binding fragment having two heavy chains and two light chains.
  • heavy chain antibody also known as “heavy chain-only antibody” or “HCAb” refers to a functional antibody, which comprises two heavy chains, but lacks two light chains usually found in 4-chain antibodies.
  • Camelid animals (such as camels, llamas, or alpacas) are known to produce HCAbs.
  • an "isolated antibody” refers to an antibody that is substantially free of other antibodies having different antigenic specificities.
  • An isolated antibody that binds specifically to an antigen can, however, have cross-reactivity to other antigens, such as homologous antigens from other species.
  • an isolated antibody can be substantially free of other cellular material and/or chemicals.
  • Antibody fragments comprise a portion of an antibody, preferably the antigen binding or variable region of the antibody.
  • Examples of antibody fragments include VHHs, single-domain antibodies, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see US Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • the term “constant domain” refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site.
  • the constant domain contains the C H 1, C H 2 and C H 3 domains (collectively, C H ) of the heavy chain and the CHL (or CL) domain of the light chain.
  • binding typically is a binding with an affinity corresponding to a K D of about 10 6 M or less, e.g.
  • the amount with which the K D of binding is lower is dependent on the K D of the antibody, so that when the K D of the antibody is very low, then the amount with which the K D of binding to the antigen is lower than the K D of binding to a non-specific antigen may be at least 10,000-fold (that is, the antibody is highly specific).
  • K D refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Affinity, as used herein, and K D are inversely related, that is that higher affinity is intended to refer to lower K D , and lower affinity is intended to refer to higher K D .
  • a “CDR” refers to one of three hypervariable regions (H1, H2, or H3) within the non- framework region of the immunoglobulin (Ig or antibody) VH P-sheet framework, or one of three hypervariable regions (L1, L2, or L3) within the non-framework region of the antibody VL ⁇ -sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences.
  • CDR regions are well known to those skilled in the art and have been defined by, for example, Kabat as the regions of most hypervariability within the antibody variable (V) domains. Kabat et al., J. Biol. Chem. 1977, 252, 6609-6616; Kabat, Adv. Protein Chem. 1978, 32, 1-75. CDR region sequences also have been defined structurally by Chothia as those residues that are not part of the conserved P-sheet framework, and thus are able to adapt different conformations. Chothia and Lesk, J. Mol. Biol. 1987, 196, 901-917. Both terminologies are well recognized in the art. CDR region sequences have also been defined by AbM, Contact and IMGT.
  • Fc region herein is used to define a C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is often defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxy 1-terminus thereof.
  • the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.
  • variable region refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen.
  • the variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs).
  • FRs conserved framework regions
  • HVRs hypervariable regions
  • antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
  • hypervariable region refers to each of the regions of an antibody variable domain which are hypervariable in sequence (“complementarity determining regions” or “CDRs”) and/or form structurally defined loops (“hypervariable loops”) and/or contain the antigen-contacting residues (“antigen contacts”).
  • CDRs complementarity determining regions
  • hypervariable loops form structurally defined loops
  • antigen contacts antigen contacts
  • 4-chain antibodies and antigen- binding antibody fragments thereof comprise six HVRs: three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3).
  • heavy-chain antibodies comprise three HVRs (HVR1, HVR2, HVR3).
  • HVRs for 4-chain antibodies and antigen-binding antibody fragments thereof herein include: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol.
  • 262 732-745 (1996)); and (d) combinations of (a), (b), and/or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).
  • amino acid residues of a single-domain antibody can be numbered according to the general numbering for VH domains given by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, Md., Publication No. 91), as applied to VHH domains from Camelids in the article of Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240 (1-2): 185-195.
  • FR1 of a VHH comprises the amino acid residues at positions 1-30
  • CDR1 of a VHH comprises the amino acid residues at positions 31-35
  • FR2 of a VHH comprises the amino acids at positions 36-49
  • CDR2 of a VHH comprises the amino acid residues at positions 50-65
  • FR3 of a VHH comprises the amino acid residues at positions 66-94
  • CDR3 of a VHH comprises the amino acid residues at positions 95-102
  • FR4 of a VHH comprises the amino acid residues at positions 103-113.
  • the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering).
  • CDR residues and other residues in the variable domain are numbered herein according to Kabat et al.
  • cassette refers to a sequence of DNA carrying, and capable of directing the expression of, one or more genes of interest between one or more sets of restriction sites. It can be transferred from one DNA sequence (usually a vector) to another by “cutting” the fragment out using restriction enzymes and “pasting” it back into the new context (such as a viral genome).
  • the DNA fragment is operatively associated with expression control sequence elements which provide for the proper transcription and translation of the target nucleic acid sequence(s) (genes).
  • sequence elements may include a promoter and a polyadenylation signal.
  • a sequence “encoding” an expression product such as a polypeptide, is a minimum nucleotide sequence that, when expressed, results in the production of that polypeptide.
  • exogenous refers to a combination of elements not naturally occurring.
  • an “exogenous gene” refers to a gene to be introduced to the genome of a virus, wherein that gene is not normally found in the genome of the virus or is a homolog of a gene expressed in the virus from a different species (e.g., the bovine herpes virus UL49.5 gene, which encodes for a TAP inhibitor, is exogenous when inserted into a viral genome that does not natively encode UL49.5).
  • herpes simplex virus refers to members of the Herpesviridae family.
  • Herpes simplex virus 1 and 2 also known by their taxonomical names Human alphaherpesvirus I and Human alphaherpesvirus 2
  • HSV-1 and HSV-2 also known by their taxonomical names Human alphaherpesvirus I and Human alphaherpesvirus 2
  • Human alphaherpesvirus I and Human alphaherpesvirus 2 are two members of the human Herpesviridae family, a set of viruses that produce viral infections in the majority of humans.
  • Percent (%) amino acid sequence identity or “homology” with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.
  • the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087.
  • the ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California.
  • the ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • the term “specific binding” or “specifically binds” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a K D for the target of at least about 10 -4 M, alternatively at least about 10 -5 M, alternatively at least about 10 -6 M, alternatively at least about 10 -7 M, alternatively at least about 10 -8 M, alternatively at least about 10 -9 M, alternatively at least about 10 -10 M, alternatively at least about 10 -11 M, alternatively at least about 10 -12 M, or greater.
  • the term “specific binding” refers to binding where a molecule binds a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.
  • K D can be determined by methods known in the art, such as ELISA, surface plasmon resonance (SPR), fluorescence activated cell sorting (FACS) analysis, or radioimmunoprecipitation (RIA).
  • Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity.
  • specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target.
  • An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non- human primates such as rhesus and cynomolgus monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.
  • a "cancer” refers herein to solid tumors.
  • Treatment refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of curing, reversing, alleviating, ameliorating, inhibiting, slowing down, or preventing the onset, progression, development, severity, or recurrence of a symptom, complication, condition, or biochemical indicia associated with a disease.
  • the disease is cancer.
  • the phrase "pharmaceutically acceptable” indicates that the substance or composition must be compatible chemically and/or toxicologically, with the other ingredients comprising a formulation, and/or the mammal being treated therewith.
  • the term “no more than” refers to an amount that is less than or equal to. This may be an amount in integers. For example, no more than two lesions can refer to 0, 1, or 2 lesions.
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • Description of endpoints includes ranges between all endpoints disclosed. For example description of 1, 2, or 3 includes the ranges 1-2, 2-3 and 1-3.
  • Immunotherapy of cancer with oncolytic viruses is an emerging and maturing treatment modality which uses replication-competent viruses that selectively infect and damage tumor cells and may also, preferably, induce an immunological response which can control both the target tumor and distal tumors.
  • Each species of oncolytic virus has a different cellular tropism, which helps determine which tissues are preferentially infected. Engineering of the virus can expand, restrict, or modulate this host range.
  • a variety of species of virus have been investigated for use in oncolytic therapies, including those derived from HSV, vaccinia, and reovirus.
  • the present application provides oncolytic viruses that are effective for treating cancer.
  • oncolytic viruses include those derived from a herpes simplex virus, a vaccinia virus, an adenovirus, a reovirus, or a vesicular stomatitis virus.
  • the oncolytic virus (such as an oncolytic HSV) preferentially triggers an immune response that results in killing of tumor cells.
  • the virus “preferentially kills” tumor cells when certain infectious doses of the virus are more likely to kill tumor cells than neighboring healthy cells (such as at least two times more likely to kill tumor cells than neighboring healthy cells at a given dose).
  • the oncolytic virus expresses one or more payload proteins described below.
  • the oncolytic virus induces an immune response to the tumor, which, in some embodiments, causes tumor cells at sites distal to the site of infection to be killed.
  • the oncolytic virus is capable of evading an individual’s immune system after administration to the individual. As used herein, evading the individual’s immune system means that the oncolytic virus is able to preferentially replicate in tumor cells.
  • the oncolytic viruses provided herein are more sensitive to an innate antiviral response than a wild-type virus, enabling preferential replication in tumor cells.
  • the oncolytic viruses provided herein have an intermediate resistance to interferon.
  • the present disclosure pertains to an oncolytic herpes simplex virus (HSV).
  • HSV herpes simplex virus
  • the oncolytic HSV is derived from HSV-1.
  • the present disclosure pertains to a monotherapy comprising administering the HSV-1 virus intratumorally to an individual for treating solid tumors.
  • the present disclosure pertains to a combination therapy comprising administering the HSV-1 virus intratumorally and administering an antibody that binds to PD-1 to an individual for treating solid tumors.
  • the oncolytic HSV comprises one or more expression cassettes described herein.
  • the oncolytic HSV expresses one or more payload proteins described herein.
  • the oncolytic HSV lacks one or more native HSV genes. In some embodiments, the oncolytic HSV lacks one or both copies of ⁇ 34.5. In some embodiments, the oncolytic HSV does not express one or more native HSV proteins, such as US 12. In some embodiments, the oncolytic HSV expresses one or more additional copies of a native HSV protein, such as US11. In some embodiments, the oncolytic HSV expresses a native HSV protein in a different temporal order, such as expressing immediate-early US11. The oncolytic HSV may be a component of a pharmaceutical composition described herein.
  • the oncolytic HSV may be administered to individual according to the methods described herein (such as the methods of treatment described herein).
  • the oncolytic HSV preferentially triggers an immune response that results in killing of tumor cells compared to the wild-type HSV from which it is derived.
  • the oncolytic HSV is capable of triggering an immune response that triggers killing tumor cells at one or more sites distal to a target site.
  • the oncolytic virus (such as an oncolytic HSV), or the gene cassette otherwise described herein, comprises one or more genes encoding one or more payload molecules.
  • the payload molecules are generally intended to enhance the therapeutic effectiveness of the oncolytic virus (such as an oncolytic HSV).
  • a payload molecule may promote an immune response (e.g., against the tumor target) or may enhance the cytotoxicity of the oncolytic virus.
  • an oncolytic virus (such as an oncolytic HSV), or an expression cassette otherwise described herein, comprises a polynucleotide encoding interleukin 12 (IL- 12).
  • IL-12 is a heterodimeric protein comprising two subunits: p35 and p40.
  • the native p35 subunit is linked to the p40 subunit by a disulfide bond.
  • the human and mouse p40 subunits are 70% identical, while the p35 subunits share 60% amino acid sequence homology.
  • the p35 and p40 subunits may function in receptor binding and signal transduction, respectively (Zou, J. J., et al. (1995).
  • IL-12 is normally secreted by antigen-presenting cells, such as macrophages and dendritic cells.
  • Biologically active IL- 12 (comprising both subunits in a heterodimer) functions to differentiate naive T cells into Th1 cells, promote cytotoxic activity of NK cells and T cells, and block angiogenesis.
  • the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises a polynucleotide encoding the p35 subunit of IL- 12 and/or a polynucleotide encoding the p40 subunit of IL- 12.
  • the p35 subunit and/or p40 subunit of IL-12 is human.
  • the p35 subunit and/or p40 subunit of IL- 12 is murine.
  • the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises a polynucleotide encoding an IL- 12 heterodimer comprising a p35 subunit and a p40 subunit.
  • the IL- 12 heterodimer comprises a polypeptide comprising a p35 subunit of IL- 12 and a p40 subunit of IL- 12 connected by a peptide linker.
  • the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises a polynucleotide encoding a human p35 subunit of IL- 12 and/or a polynucleotide encoding a human p40 subunit of IL- 12.
  • the human p35 subunit comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:1.
  • the human p40 subunit comprises the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:2.
  • the human p40 subunit comprises the amino acid sequence of SEQ ID NO:9, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 9.
  • the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises a polynucleotide encoding an IL- 12 heterodimer comprising a human p35 subunit and a human p40 subunit.
  • the IL- 12 heterodimer comprises a polypeptide comprising a human p35 subunit of IL- 12 and a human p40 subunit of IL- 12 connected by a peptide linker.
  • the peptide linker comprises an amino acid sequence comprising glycine and serine residues.
  • the peptide linker comprises the amino acid sequence of SEQ ID NO:3.
  • the peptide linker comprises the amino acid sequence of SEQ ID NO: 7.
  • the IL-12 heterodimer comprises the amino acid sequence of SEQ ID NO:4In some embodiments, the IL-12 heterodimer comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 10.
  • the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises a polynucleotide encoding a murine p35 subunit of IL-12 and/or a polynucleotide encoding a murine p40 subunit of IL-12.
  • the murine p35 subunit comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about
  • the murine p40 subunit comprises the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about
  • the murine p40 subunit comprises the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having any of at least about 80%, at least about
  • the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises a polynucleotide encoding an IL- 12 heterodimer comprising a murine p35 subunit and a murine p40 subunit.
  • the IL- 12 heterodimer comprises a polypeptide comprising a murine p35 subunit of IL- 12 and a murine p40 subunit of IL- 12 connected by a peptide linker.
  • the peptide linker comprises an amino acid sequence comprising glycine and serine residues. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:3.
  • the peptide linker comprises the amino acid sequence of SEQ ID N0:7
  • the IL-12 heterodimer comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 12.
  • Cluster of differentiation 40 is a costimulatory polypeptide expressed on numerous cell types, from antigen presenting cells (APCs) to epithelial cells. It is additionally present on various cancer cells.
  • CD40 agonist also known as cluster of differentiation 154 (CD154), comprises 261 amino acids and is a type II membrane glycopolypeptide that is expressed on the surface of activated T cells. Native CD40 agonist promotes B cell maturation. It is additionally essential for immunoglobulin class switching, as lack of CD40 agonist is associated with hyper IgM syndrome.
  • CD40 agonist exists as a membrane-bound form, in which the extracellular domain forms a homotrimer, and a proteolytically-cleaved, soluble form, which has been shown to be biologically active.
  • an oncolytic virus comprising a polynucleotide encoding a CD40 agonist.
  • an oncolytic virus (such as an oncolytic HSV), or an expression cassette otherwise described herein, comprises a polynucleotide encoding CD40 agonist.
  • the CD40 agonist is a CD40 ligand.
  • the CD40 agonist comprises a CD40 ligand ectodomain.
  • the CD40 agonist is a trimer of three single-chain trimeric CD40 ligand ectodomains.
  • each of the three single-chain trimeric CD40 ligand ectodomains is fused to a trimerization motif, e.g., to direct formation of the trimer of three single-chain trimeric CD40 ligand ectodomains.
  • each of the three single-chain trimeric CD40 ligand ectodomains is fused to an Fc region, e.g., to direct formation of the trimer of three single-chain trimeric CD40 ligand ectodomains.
  • said Fc region is an IgG Fc region e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region.
  • said Fc region comprises one or more amino acid substitutions, insertions, or deletions that disfavor binding of said Fc region to another Fc region, such as an IgG Fc region, e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region.
  • said Fc region comprises a substitution of the IgG interaction domain with an IgA interaction domain.
  • each of the three single-chain trimeric CD40 ligand ectodomains is bivalent.
  • the CD40 agonist is an agonist antibody.
  • the CD40 agonist comprises a human CD40 ligand ectodomain.
  • the human CD40 ligand ectodomain comprises the amino acid sequence set forth in SEQ ID NO:20, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 20.
  • the CD40 agonist is a trimer of three single-chain trimeric human CD40 ligand ectodomains.
  • the single-chain trimeric human CD40 ligand ectodomains comprise a polypeptide comprising three human CD40 ligand ectodomains connected by peptide linkers.
  • the single-chain trimeric human CD40 ligand ectodomain polypeptide comprises a first human CD40 ligand ectodomain connected by a peptide linker to a second human CD40 ligand ectodomain which is connected by a peptide linker to a third human CD40 ligand ectodomain.
  • the peptide linker comprises glycine and serine residues. In some embodiments, the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 22.
  • the CD40 agonist comprises a trimerization motif operably linked to each of the three single-chain trimeric CD40 ligand ectodomains. In some embodiments, the trimerization motif is a T4 fibritin trimerization motif. In some embodiments, the T4 fibritin trimerization motif comprises the amino acid sequence set forth in SEQ ID NO:21. In some embodiments, the trimerization motif is linked to each of the three single-chain trimeric CD40 ligand ectodomains by a peptide linker.
  • the peptide linker connecting the trimerization motif to each of the three single-chain trimeric CD40 ligand ectodomains comprises an amino acid sequence comprising of glycine and/or serine residues. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO:23In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO:27. In some embodiments, the CD40 agonist further comprises a signal peptide sequence operably linked to each of the three single-chain trimeric CD40 ligand ectodomains. In some embodiments, the signal peptide sequence comprises the amino acid sequence of SEQ ID NO:24.
  • the CD40 agonist comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:25, or an amino acid sequence having any of about at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:25.
  • the CD40 agonist comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having any of about at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:30.
  • the CD40 agonist forms or is a trimer comprising three polypeptides comprising the amino acid sequence of SEQ ID NO:25, or an amino acid sequence having any of about at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:25.
  • the CD40 agonist forms or is a trimer comprising three polypeptides comprising the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having any of about at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:30.
  • the CD40 agonist comprises a murine CD40 ligand ectodomain.
  • the murine CD40 ligand ectodomain comprises the amino acid sequence set forth in SEQ ID NO:26, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 26.
  • the CD40 agonist is a trimer of three single-chain trimeric murine CD40 ligand ectodomains.
  • the single-chain trimeric murine CD40 ligand ectodomains comprise a polypeptide comprising three murine CD40 ligand ectodomains connected by peptide linkers.
  • the single-chain trimeric murine CD40 ligand ectodomain polypeptide comprises a first murine CD40 ligand ectodomain connected by a peptide linker to a second murine CD40 ligand ectodomain which is connected by a peptide linker to a third murine CD40 ligand ectodomain.
  • the peptide linker comprises glycine and serine residues. In some embodiments, the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 22.
  • the CD40 agonist comprises a trimerization motif operably linked to each of the three single-chain trimeric CD40 ligand ectodomains. In some embodiments, the trimerization motif is a T4 fibritin trimerization motif. In some embodiments, the T4 fibritin trimerization motif comprises the amino acid sequence set forth in SEQ ID NO:21. In some embodiments, the trimerization motif is linked to each of the three single-chain trimeric CD40 ligand ectodomains by a peptide linker.
  • the peptide linker connecting the trimerization motif to each of the three single-chain trimeric CD40 ligand ectodomains comprises an amino acid sequence comprising of leucine, glycine, and/or serine residues. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO:23. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO:27. In some embodiments, the CD40 agonist further comprises a signal peptide sequence operably linked to each of the three single-chain trimeric CD40 ligand ectodomains. In some embodiments, the signal peptide sequence comprises the amino acid sequence of SEQ ID NO: 24.
  • the CD40 agonist comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 28
  • the CD40 agonist comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:29, or an amino acid sequence having any of about at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:29.
  • the CD40 agonist forms a trimer comprising three polypeptides comprising the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:29.
  • Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4 or CTLA-4), also known as cluster of differentiation 152 (CD 152), is a polypeptide receptor that functions as an immune checkpoint and downregulates immune responses.
  • the polypeptide contains an extracellular V-like domain, a transmembrane domain, and a cytoplasmic tail. Alternate isoforms have been characterized.
  • CTLA-4 is constitutively expressed in regulatory T cells, but is only upregulated in conventional T cells after activation, and contributes to the inhibitory function of regulatory T cells.
  • CTLA-4 binds to CD80 and CD86, also known as B7-1 and B7-2 respectively, on APCs in order to induce its inhibitory function to T cells.
  • an oncolytic virus (such as an oncolytic HSV), or an expression cassette otherwise described herein, comprises a polynucleotide encoding a CTLA-4 binding protein.
  • the CTLA-4 binding protein is a CTLA-4 antagonist. Lor example, in some instances, the CTLA-4 binding protein inhibits the interaction between CTLA-4 and one or more CTLA-4 ligands, such as CD80 and/or CD86. In some embodiments, the CTLA-4 binding protein specifically binds to human CTLA-4, murine CTLA-4, or both human and murine CTLA- 4.
  • the CTLA-4 binding protein is an anti-CTLA-4 antibody or antigen binding fragment thereof.
  • the anti-CTLA-4 antibody or antigen binding fragment thereof specifically binds to human CTLA-4, murine CTLA-4, or both human and murine CTLA-4.
  • the anti-CTLA-4 antibody or antigen binding fragment is bivalent.
  • the anti-CTLA-4 antibody or antigen binding fragment comprises an Fc region, such as an active Fc region.
  • the anti-CTLA-4 antibody or antigen binding fragment comprises an IgG1, IgG2, IgG3, or IgG4 constant domain, e.g., a human or mouse IgG1, IgG2, IgG3, or IgG4 constant domain.
  • the anti-CTLA-4 antibody or antigen binding fragment thereof comprises a single-chain variable fragment (scFv).
  • the anti-CTLA-4 scFv is fused to the N-terminus of an IgG1, IgG2, IgG3, or IgG4 constant domain, e.g., a human or mouse IgG1, IgG2, IgG3, or IgG4 constant domain.
  • the anti-CTLA-4 antibody or antigen binding fragment thereof comprises an anti-CTLA-4 VHH, e.g., a camelid antibody comprising an anti-CTLA-4 VHH.
  • the anti-CTLA-4 VHH is fused to the heavy chain of an IgG1, IgG2, IgG3, or IgG4 Fc, e.g., a human or mouse IgG1, IgG2, IgG3, or IgG4 Fc.
  • the anti-CTLA-4 antibody or antigen binding fragment thereof, such as the anti-CTLA-4 scFv specifically binds to human CTLA-4.
  • the anti- CTLA-4 scFv is fused to the N-terminus of a IgG1 constant domain, e.g., a human IgG1 constant domain.
  • the human IgG1 is a variant human IgG1 comprising a C220S substitution, wherein the numbering of the residues is according to EU numbering.
  • the human IgG1 is a G1m(17) IgG1.
  • the anti-CTLA-4 antibody causes depletion of regulatory T (Treg) cells.
  • the anti-CTLA-4 antibody or antigen binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises one or more of: (a) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:40; (b) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO:41; and (c) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:42; and/or wherein the VL comprises one or more of: (a) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO:43; (b) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO:44; and (c) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO:45.
  • VH variable heavy chain
  • VL variable light chain
  • the anti-CTLA-4 antibody or antigen binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:46, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:46; and/or a VL comprising the amino acid sequence set forth in SEQ ID NO:47, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:46; and/or a
  • the anti-CTLA-4 antibody or antigen binding fragment thereof comprises an IgG1 constant domain comprising the amino acid sequence set forth in SEQ ID NO:48, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:48.
  • the heavy chain of the CTLA- 4 antibody comprises the amino acid sequence set forth in SEQ ID NO48, with or without the C terminal lysine.
  • the anti-CTLA-4 antibody or antigen binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:60, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:60.
  • the anti-CTLA-4 antibody or antigen binding fragment thereof comprises a signal peptide sequence comprising the amino acid sequence set forth in SEQ ID NO:49.
  • the anti-CTLA-4 antibody or antigen binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 50, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 50.
  • the anti-CTLA-4 antibody or antigen binding fragment thereof such as the anti-CTLA-4 VHH, specifically binds to murine CTLA-4.
  • the anti- CTLA-4 VHH is fused to the heavy chain of a murine IgG2a Fc.
  • the anti-CTLA-4 antibody or antigen binding fragment thereof comprises a variable heavy chain (VH), wherein the VH comprises one or more of: (a) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:51; (b) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 52; and (c) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 53.
  • VH variable heavy chain
  • the anti- CTLA-4 antibody or antigen binding fragment thereof comprises a VH, wherein the VH comprises a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 51 , a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 52, and a CDR- H3 comprising the amino acid sequence set forth in SEQ ID NO: 53.
  • the anti-CTLA-4 antibody or antigen binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 54, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 54.
  • the anti-CTLA-4 antibody or antigen binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 58, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 58.
  • the anti-CTLA-4 antibody or antigen binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 59, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about
  • the anti-CTLA-4 antibody or antigen binding fragment thereof comprises a signal peptide sequence comprising the amino acid sequence set forth in SEQ ID NO: 55.
  • the anti-CTLA-4 antibody or antigen binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 56.
  • the anti-CTLA-4 antibody or antigen binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:57.
  • the oncolytic herpes simplex type 1 (HSV-1) virus comprising a polynucleotide encoding an antibody to human cytotoxic T lymphocyte-associated protein 4 ( ⁇ CTLA4) is administered to an individual, and an antibody to human cytotoxic T lymphocyte-associated protein 4 ( ⁇ CTLA4) can further be administered to the individual.
  • the antibody to human cytotoxic T lymphocyte-associated protein 4 ( ⁇ CTLA4) encoded within the oncolytic virus and antibody to human cytotoxic T lymphocyte-associated protein 4 ( ⁇ CTLA4) administered systemically to the individual are the same antibody.
  • the antibody to human cytotoxic T lymphocyte-associated protein 4 ( ⁇ CTLA4) encoded within the oncolytic virus and antibody to human cytotoxic T lymphocyte-associated protein 4 ( ⁇ CTLA4) administered systemically to the individual are the different antibodies.
  • an oncolytic virus (such as an oncolytic HSV), or an expression cassette otherwise described herein, comprises a polynucleotide encoding a fms-like tyrosine kinase 3 ( FLT3) ligand (FLT3L).
  • FLT3L is a growth and differentiation factor that enhances and expands dendritic cells (DCs) as well as recruits DCs to the tumor microenvironment.
  • the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises a polynucleotide encoding a human FLT3L.
  • the human FLT3L comprises the amino acid sequence of SEQ ID NO: 72, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 72.
  • the human FLT3L comprises a signal peptide directing secretion to the plasma membrane.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 70.
  • the human FLT3L comprises the amino acid sequence of SEQ ID NO:71, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 71.
  • the FLT3L e.g., the human FLT3L
  • the human FLT3L is proteolytically processed into soluble FLT3L.
  • the soluble FLT3L forms a homodimer.
  • an oncolytic virus (such as an oncolytic HSV), or an expression cassette otherwise described herein, comprises one or more polynucleotides encoding a US 11 protein, such as a US11 protein from an HSV, e.g., an HSV-1 or HSV-2.
  • the protein kinase R (PKR) pathway is a component of the host cellular innate anti-viral response. PKR becomes activated in response to binding double-stranded RNA (dsRNA), a byproduct of viral replication, leading to phosphorylation and inactivation of eukaryotic translation initiation Factor 2 Subunit 1 (eIF2 ⁇ ), a translation initiation factor. Phosphorylated eIF2 ⁇ prevents translation initiation, a cellular defense mechanism aimed at blocking the production of viral proteins.
  • dsRNA double-stranded RNA
  • eIF2 ⁇ eukaryotic translation initiation Factor 2 Subunit 1
  • the US11 protein is believed to bind and sequester dsRNA, preventing the activation of the PKR pathway in host cells, and enabling enhanced viral replication.
  • the US 11 protein comprises the amino acid sequence of SEQ ID NO:80, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 80.
  • the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises a polynucleotide encoding a US11 protein, wherein the polynucleotide comprises a native US11 gene nucleotide sequence, e.g., from an HSV, such as an HSV-1 or an HSV-2.
  • the native US11 gene is a native US11 late gene, wherein the US11 protein is expressed in the late stage of viral replication.
  • the native US11 late gene is under the control of the endogenous US11 promoter, e.g., from an HSV, such as an HSV-1 or an HSV-2.
  • the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises a polynucleotide comprising a variant US11 gene.
  • the variant US 11 gene is codon optimized for expression of the US 11 protein in human cells.
  • the variant US11 gene encodes a wild type US11 protein, e.g., from an HSV, such as an HSV-1 or an HSV-2.
  • the variant US11 gene comprises the nucleotide sequence of SEQ ID NO:204, or a nucleotide sequence having any of at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the nucleotide sequence of SEQ ID NO:204.
  • the variant US 11 gene is operably linked to a promoter.
  • the promoter directs immediate early expression of the US 11 protein during viral replication.
  • the promoter is an endogenous US 12 promoter from an HSV, such as HSV-1 or HSV-2, or a portion thereof.
  • the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises both a polynucleotide encoding a US11 protein and comprising a native US11 gene nucleotide sequence, e.g., as described above; and a polynucleotide comprising a variant US11 gene, e.g., as described above.
  • an oncolytic virus (such as an oncolytic HSV), or an expression cassette otherwise described herein, comprises a polynucleotide encoding a transporter associated with antigen processing (TAP) inhibitor, such as a viral TAP inhibitor.
  • TAP antigen processing
  • viral TAP inhibitors prevent TAP from transporting peptides into the lumen of the endoplasmic reticulum, thus impairing peptide loading onto major histocompatibility complex (MHC) Class I molecules for display at the cell surface (Verweij et al. Viral inhibition of the transporter associated with antigen processing (TAP): A striking example of functional convergent evolution. PLoS Pathog. 2015; 11(4): el 004743).
  • TAP inhibition disrupts the transport of newly-expressed MHC molecules to the cell surface, this does not block pre-existing antigen display.
  • TAP inhibition by a TAP inhibitor can prevent the display of viral antigens on the cell surface, preventing premature clearance of infected cells and enabling virus persistence throughout multiple rounds of virus replication.
  • the TAP inhibitor is derived from herpes virus 1 or herpes virus 2. In some embodiments, the TAP inhibitor is derived from bovine herpes virus 1. In some embodiments, the TAP inhibitor is any of UL49.5, US6, or ICP47. In some embodiments, the TAP inhibitor is UL49.5.
  • the TAP inhibitor comprises the amino acid sequence of SEQ ID NO:83, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 83.
  • the TAP inhibitor further comprises a signal peptide sequence.
  • the signal peptide sequence comprises the amino acid sequence of SEQ ID NO: 81.
  • the TAP inhibitor comprises the amino acid sequence of SEQ ID NO: 82, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 82.
  • the TAP inhibitor is expressed during the immediate early phase of viral replication, i.e., it is expressed as an immediate early gene.
  • the polynucleotide encoding the TAP inhibitor is expressed under the control of an immediate early promoter, such as a CMV promoter, e.g., an hCMV promoter.
  • one or more expression cassettes comprising a polynucleotide encoding IL- 12, a polynucleotide encoding a CD40 agonist, a polynucleotide encoding a CTLA-4 binding protein, a polynucleotide encoding an FLT3 ligand (FLT3L), or any combination thereof.
  • V-A Expression cassettes encoding IL- 12, a CD40 agonist, and/or a CTLA-4 binding protein
  • expression cassettes comprising a polynucleotide encoding IL- 12, a polynucleotide encoding a CD40 agonist, and/or a polynucleotide encoding a CTLA-4 binding protein.
  • the expression cassettes of the disclosure comprise a promoter operably linked to each of the polynucleotide encoding IL- 12, the polynucleotide encoding the CD40 agonist, and/or the polynucleotide encoding the CTLA-4 binding protein.
  • Any suitable promoter may be used in the cassettes of the disclosure, so long as the promoter drives expression of the associated polynucleotide.
  • Exemplary and non-limiting promoters that may be used include the human cytomegalovirus (hCMV) promoter, the murine cytomegalovirus (mCMV) promoter, the Aotine betaherpesvirus 1 (AoHV 1) promoter, the CAG promoter, a CMV hybrid promoter, the EF1a promoter, the MMLV 5’ long terminal repeat (LTR) from the Moloney murine leukemia virus promoter (i.e., the MMLV promoter), the Pbidir3 promoter, and a native HSV promoter sequence, such as the HSV-1 or HSV-2 US 12 promoter, or the HSV-1 or HSV-2 US11 promoter.
  • hCMV human cytomegalovirus
  • mCMV murine cytomegalovirus
  • AoHV 1 Aotine betaherpesvirus 1
  • CAG CAG promoter
  • CMV hybrid promoter the CAG promoter
  • EF1a promoter the EF1a promoter
  • the expression cassettes of the disclosure comprise a polyadenylation signal operably linked to each of the polynucleotide encoding IL- 12, the polynucleotide encoding the CD40 agonist, and/or the polynucleotide encoding the CTLA-4 binding protein. Any suitable polyadenylation signal may be used in the cassettes of the disclosure.
  • Exemplary and non-limiting polyadenylation signals that may be used include the simian vacuolating virus 40 polyA (SV40pA), the human beta globin polyA (hBGpA), the human growth hormone polyA(hGH polyA), the rabbit beta globin polyA (rBGpA), a bovine growth hormone polyadenylation (BGHpA), a polyA derived from the human GAPDH gene, and a native HSV polyA sequence, such as the US10-12 polyA or the US9-10 polyA from HSV-1 or HSV-2.
  • SV40pA simian vacuolating virus 40 polyA
  • hBGpA human beta globin polyA
  • rBGpA human growth hormone polyA
  • BGHpA bovine growth hormone polyadenylation
  • a polyA derived from the human GAPDH gene such as the US10-12 polyA or the US9-10 polyA from HSV-1 or HSV-2.
  • the expression cassettes of the disclosure may comprise any suitable promoter and/or polyadenylation signal known in the art or described herein operably linked to any of the polynucleotide encoding IL- 12, the polynucleotide encoding the CD40 agonist, and/or the polynucleotide encoding the CTLA-4 binding protein.
  • the expression cassettes of the disclosure comprise an RNA Polymerase II transcriptional pause signal positioned after each of the polynucleotide encoding IL- 12, the polynucleotide encoding the CD40 agonist, and/or the polynucleotide encoding the CTLA-4 binding protein.
  • Any suitable RNA Polymerase II transcriptional pause signal may be used in the cassettes of the disclosure.
  • Exemplary and non-limiting RNA polymerase II transcriptional pause signals include the human complement C2 protein terminator (C2) and the human Gastrin terminator (hGT).
  • an expression cassette of the disclosure comprises, in order, the polynucleotide encoding the CTLA-4 binding protein, the polynucleotide encoding the CD40 agonist, and the polynucleotide encoding the IL- 12.
  • the polynucleotide encoding the CTLA-4 binding protein and the polynucleotide encoding the IL- 12 are in the same orientation in the expression cassette, and the polynucleotide encoding the CD40 agonist is in the reverse orientation relative to the polynucleotide encoding the CTLA-4 binding protein and the polynucleotide encoding the IL- 12.
  • the polynucleotide encoding the CTLA-4 binding protein is operably linked to a promoter, such as any suitable promoter known in the art or described herein.
  • the promoter is an mCMV promoter.
  • the expression cassette comprises a polyadenylation signal operably linked to the polynucleotide encoding the CTLA-4 binding protein, such as any suitable polyadenylation signal known in the art or described herein.
  • the polyadenylation signal is a polyA derived from the human GAPDH gene.
  • the expression cassette further comprises a Kozak sequence positioned between the promoter and the polynucleotide encoding the CTLA-4 binding protein
  • the expression cassette further comprises an RNA polymerase II transcriptional pause signal positioned after the polyadenylation signal, such as any suitable RNA polymerase II transcriptional pause signal known in the art or described herein.
  • the RNA polymerase II transcriptional pause signal is a C2 RNA polymerase II transcriptional pause signal.
  • the encoded CTLA-4 binding protein is any of the CTLA-4 binding proteins described herein, e.g., in Section III-C, above.
  • the polynucleotide encoding the CD40 agonist is operably linked to a promoter, such as any suitable promoter known in the art or described herein.
  • the promoter is the AOHV1 promoter.
  • the expression cassette further comprises a polyadenylation signal operably linked to the polynucleotide encoding the CD40 agonist, such as any suitable polyadenylation signal known in the art or described herein.
  • the polyadenylation signal is a hBGpA.
  • the expression cassette further comprises a Kozak sequence positioned between the promoter and the polynucleotide encoding the CD40 agonist.
  • the expression cassette further comprises an RNA polymerase II transcriptional pause signal positioned after the polyadenylation signal, such as any suitable RNA polymerase II transcriptional pause signal known in the art or described herein.
  • the RNA polymerase II transcriptional pause signal is the hGT RNA polymerase II transcriptional pause signal.
  • the encoded CD40 agonist is any of the CD40 agonists described herein, e.g., in Sections III-B or V, herein.
  • the polynucleotide encoding the CD40 agonist is in the reverse orientation within the expression cassette relative to the polynucleotide encoding the IL- 12 and the polynucleotide encoding the CTLA-4 binding protein.
  • the polynucleotide encoding IL- 12 is operably linked to a promoter, such as any suitable promoter known in the art or described herein.
  • the promoter is the MMLV promoter
  • the expression cassette further comprises a polyadenylation signal positioned after the polynucleotide encoding IL- 12, such as any suitable polyadenylation signal known in the art or described herein.
  • the polyadenylation signal is the US 10- 12 poly A or the US 9- 10 polyA from HSV, such as from HSV- 1 or HSV-2.
  • the US 10- 12 polyA comprises the nucleotide sequence of a native HSV-1 or HSV-2 US 10- 12 poly A.
  • the expression cassette further comprises a Kozak sequence positioned between the promoter and the polynucleotide encoding IL- 12.
  • an expression cassette of the disclosure further comprises a polynucleotide encoding a US 10 protein and/or a polynucleotide encoding a US 11 protein; or a polynucleotide encoding a US11 protein and a US 10 protein.
  • an expression cassette of the disclosure comprises a polynucleotide encoding a US 10 protein and/or a polynucleotide encoding a US11 protein.
  • the polynucleotide encoding the US 11 protein is operably linked to a promoter, such as any suitable promoter known in the art or described herein.
  • the promoter is an endogenous US11 promoter from an HSV, such as HSV-1 or HSV-2.
  • the endogenous US11 promoter directs late expression of the US11 protein during viral replication.
  • the polynucleotide encoding the US 10 protein is operably linked to a promoter, such as any suitable promoter known in the art or described herein.
  • the promoter is an endogenous US 10 promoter.
  • the expression cassette comprises a polyadenylation signal operably linked to the polynucleotide encoding the US 10 protein, such as any suitable polyadenylation signal known in the art or described herein.
  • the polyadenylation signal is a hGHpolyA.
  • the encoded US 11 protein is an HSV US 11 protein, such as an HSV-1 or HSV-2 US 11 protein.
  • the polynucleotide encoding the US 11 protein comprises a native US 11 gene.
  • the expression cassette comprises, in order, the polynucleotide encoding the US11 protein (e.g., comprising a native US11 gene) and/or the polynucleotide encoding the US 10 protein, the polynucleotide encoding the CTLA-4 binding protein, the polynucleotide that encodes the CD40 agonist, and the polynucleotide encoding the IL- 12.
  • the polynucleotides encoding the CTLA-4 binding protein, the IL- 12, the US 11 protein and/or the US 10 protein are in the same orientation in the expression cassette, and the polynucleotide that encodes the CD40 agonist is in the reverse orientation relative to the polynucleotides encoding the CTLA-4 binding protein, the IL- 12, the US 11 protein and/or the US 10 protein.
  • an expression cassette of the disclosure comprises a polynucleotide encoding a US11 protein and a US 10 protein.
  • the polynucleotide encoding the US 11 protein and the US 10 protein comprises a nucleic acid sequence encoding the US 11 protein, and a nucleic acid sequence encoding the US 10 protein.
  • at least a portion of the nucleic acid sequence encoding the US11 protein overlaps with at least a portion of the nucleic acid sequence encoding the US 10 protein.
  • the nucleic acid sequence encoding the US 11 protein is operably linked to a promoter, such as any suitable promoter known in the art or described herein.
  • the promoter is an endogenous US11 promoter from an HSV, such as HSV-1 or HSV-2. In some embodiments, the endogenous US11 promoter directs late expression of the US11 protein during viral replication.
  • the nucleic acid sequence encoding the US 10 protein is operably linked to a promoter. In some embodiments, the promoter is a native US 10 promoter from an HSV, such as HSV-1 or HSV-2. In some embodiments, the promoter is embedded within the nucleic acid sequence encoding the US11 protein. In some embodiments, the encoded US11 protein is an HSV US 11 protein, such as an HSV-1 or HSV-2 US 11 protein.
  • the expression cassette comprises a polyadenylation signal operably linked to the nucleic acid sequence encoding the US 10 protein, such as any suitable polyadenylation signal known in the art or described herein.
  • the polyadenylation signal is a hGHpolyA.
  • the polynucleotide encoding the US 11 protein and the US 10 protein comprises a native US 11 gene.
  • the expression cassette comprises, in order, the polynucleotide encoding the US11 protein and the US 10 protein (e.g., comprising a native US11 gene), the polynucleotide encoding the CTLA-4 binding protein, the polynucleotide that encodes the CD40 agonist, and the polynucleotide encoding the IL- 12.
  • the polynucleotides encoding the CTLA-4 binding protein, the IL- 12, and the US 11 and US 10 proteins are in the same orientation in the expression cassette, and the polynucleotide that encodes the CD40 agonist is in the reverse orientation relative to the polynucleotides encoding the CTLA-4 binding protein, the IL- 12, and the US11 and US 10 proteins.
  • an expression cassette of the disclosure further comprises a polynucleotide encoding a US 11 protein, wherein the polynucleotide comprises a variant US 11 gene.
  • the variant US11 gene comprises a sequence that is codon optimized for expression of the US11 protein in human cells.
  • the variant US11 gene is operably linked to a promoter, such as any suitable promoter known in the art or described herein.
  • the promoter is an endogenous US 12 promoter from an HSV, such as HSV-1 or HSV-2, or a portion thereof.
  • the endogenous US 12 promoter, or the portion thereof directs immediate early expression of the US 11 protein during viral replication.
  • the expression cassette further comprises a 5’ untranslated region (UTR) sequence positioned between the promoter and the variant US 11 gene.
  • the expression cassette further comprises a polynucleotide encoding a US 12 protein positioned after the variant US11 gene (e.g., after a stop codon in the variant US11 gene).
  • the US12 protein is from an HSV, such as HSV-1 or HSV-2.
  • the polynucleotide encoding the US 12 protein is not operably linked to a promoter. In some embodiments, the encoded US 12 protein is not expressed.
  • the expression cassette further comprises a spacer sequence and a UTR sequence positioned between the variant US 11 gene and the polynucleotide encoding the US 12 protein.
  • the expression cassette comprises, in order, the variant US11 gene; the polynucleotides encoding the US 10 and/or US 11 proteins, or the polynucleotide encoding the US 10 and US 11 proteins; the polynucleotide encoding the CTLA-4 binding protein; the polynucleotide that encodes the CD40 agonist; and the polynucleotide encoding the IL- 12.
  • the polynucleotides encoding the CTLA-4 binding protein, the IL- 12, and the US 10 and/or US11 proteins are in the same orientation in the expression cassette, and the polynucleotide that encodes the CD40 agonist is in the reverse orientation relative to the polynucleotides encoding the CTLA-4 binding protein, the IL- 12, and the US 10 and/or US11 proteins.
  • an expression cassette of the disclosure comprises, in order, a promoter (e.g., an HSV US 12 promoter) operably linked to the polynucleotide comprising a variant US 11 gene; optionally, a 5’ UTR sequence; the polynucleotide comprising the variant US 11 gene; a promoter (e.g., a native HSV US 11 promoter); the polynucleotide encoding the US 11 protein and the US10 protein; a polyadenylation signal (e.g., a hGHpA poly A) operably linked to the polynucleotide encoding the US11 protein and the US10 protein; a promoter (e.g., a CMV promoter such as an mCMV promoter) that directs expression of the polynucleotide encoding the CTLA-4 binding protein; optionally, a Kozak sequence for expression of the polynucleotide encoding the CTLA-4 binding protein;
  • a promoter e
  • an expression cassette of the disclosure comprises the nucleotide sequence of SEQ ID NO:201, or a nucleotide sequence having any of at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the nucleotide sequence of SEQ ID NO:201.
  • an expression cassette of the disclosure comprises the nucleotide sequence of SEQ ID NO:202, or a nucleotide sequence having any of at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the nucleotide sequence of SEQ ID NO:202.
  • an expression cassette of the disclosure is integrated into a genome of a virus, such as an oncolytic HSV, e.g., an oncolytic HSV-1 or oncolytic HSV-2.
  • the cassette is integrated in the US10-12 locus of an oncolytic HSV, e.g., an oncolytic HSV-1 or oncolytic HSV-2.
  • the expression cassette comprises: (i) the polynucleotide encoding IL- 12, (ii) the polynucleotide encoding the CD40 agonist, (iii) and the polynucleotide encoding the CTLA-4 binding protein, e.g., as described above.
  • the expression cassete is in the orientation relative to the internal short repeat (IRS) region of the genome, e.g., an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome, of IRS-(i)-(ii)-(iii).
  • the expression cassette further comprises polynucleotide(s) encoding a US 10 protein and/or a US 11 protein, e.g., as described above.
  • the expression cassete comprises (i) the polynucleotide encoding IL- 12, (ii) the polynucleotide encoding the CD40 agonist, (iii) the polynucleotide encoding the CTLA-4 binding protein, and (iv) the polynucleotides encoding the US 10 protein and/or US 11 protein.
  • the expression cassete is in the orientation relative to the internal short repeat (IRS) region of the genome, e.g., an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome, of IRS-(i)-(ii)-(iii)-(iv).
  • the expression cassette further comprises a polynucleotide encoding a US 10 protein and a US 11 protein, e.g., as described above.
  • the expression cassette comprises (i) the polynucleotide encoding IL- 12, (ii) the polynucleotide encoding the CD40 agonist, (iii) the polynucleotide encoding the CTLA-4 binding protein, and (iv) the polynucleotide encoding the US 10 and US 11 proteins.
  • the expression cassete is in the orientation relative to the internal short repeat (IRS) region of the genome, e.g., an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome, of IRS-(i)-(ii)-(iv).
  • the expression cassette further comprises a polynucleotide encoding a US10 protein and a polynucleotide encoding a a US11 protein, e.g., as described above.
  • the expression cassete comprises (i) the polynucleotide encoding IL- 12, (ii) the polynucleotide encoding the CD40 agonist, (iii) the polynucleotide encoding the CTLA-4 binding protein, (iv) the polynucleotide encoding the US 10 protein, and (v) the polynucleotide encoding the US 11 protein.
  • the expression cassete is in the orientation relative to the internal short repeat (IRS) region of the genome, e.g., an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome, ofIRS-(i)-(ii)-(iii)- (iv)-(v).
  • IRS internal short repeat
  • the expression cassete further comprises a polynucleotide comprising a variant US11 gene, e.g., as described above.
  • the expression cassette comprises: (i) the polynucleotide encoding IL-12, (ii) the polynucleotide encoding the CD40 agonist, (iii) the polynucleotide encoding the CTLA-4 binding protein, (iv) the polynucleotide(s) encoding the US 10 protein and/or US11 protein, or the polynucleotide encoding the US 10 and US 11 proteins, and (v) the polynucleotide comprising the variant US 11 gene.
  • the expression cassette is in the orientation relative to the internal short repeat (IRS) region of the genome, e.g., an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome, of IRS-(i)-(ii)-(iii)-(iv)-(v).
  • the expression cassette comprises a polynucleotide comprising a variant US 11 gene, e.g., as described above.
  • the expression cassette comprises: (i) the polynucleotide encoding IL-12, (ii) the polynucleotide encoding the CD40 agonist, (iii) the polynucleotide encoding the CTLA-4 binding protein, (iv) the polynucleotide encoding the US 10 protein, (v) the polynucleotide encoding the US 11 protein, (vi) the polynucleotide comprising the variant US 11 gene.
  • the expression cassette is in the orientation relative to the internal short repeat (IRS) region of the genome, e.g., an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome, of IRS-(i)-(ii)-(iii)- (iv)-(v)-(vi).
  • IRS internal short repeat
  • expression cassettes comprising a polynucleotide encoding FLT3L and/or a polynucleotide encoding a transporter associated with antigen processing (TAP) inhibitor.
  • TAP antigen processing
  • an expression cassette of the disclosure comprises a promoter operably linked to the polynucleotide encoding FLT3L and/or to the polynucleotide encoding the TAP inhibitor. Any suitable promoter may be used in the cassettes of the disclosure, so long as the promoter drives expression of the associated polynucleotide.
  • Exemplary and non-limiting promoters that may be used include the human cytomegalovirus (hCMV) promoter, the murine cytomegalovirus (mCMV) promoter, the Aotine betaherpesvirus 1 (AoHV 1) promoter, the CAG promoter, a CMV hybrid promoter, the EF1a promoter, the MMLV 5’ long terminal repeat (LTR) from the Moloney murine leukemia virus promoter (i.e., the MMLV promoter), the Pbidir3 promoter, and a native HSV promoter sequence.
  • hCMV human cytomegalovirus
  • mCMV murine cytomegalovirus
  • AoHV 1 Aotine betaherpesvirus 1
  • the expression cassette further comprises a polyadenylation signal operably linked to the polynucleotide encoding FLT3L and/or the polynucleotide encoding the TAP inhibitor. Any suitable polyadenylation signal may be used in the cassettes of the disclosure.
  • Exemplary and non-limiting polyadenylation signals include the simian vacuolating virus 40 polyA (SV40pA), the human beta globin polyA (hBGpA), the human growth hormone polyA (hGH polyA), the rabbit beta globin polyA (rBGpA), a bovine growth hormone polyadenylation (BGHpA), a polyA derived from the human GAPDH gene, and a native HSV polyA sequence.
  • SV40pA simian vacuolating virus 40 polyA
  • hBGpA human beta globin polyA
  • hGH polyA human growth hormone polyA
  • rBGpA rabbit beta globin polyA
  • BGHpA bovine growth hormone polyadenylation
  • the expression cassettes of the disclosure may comprise any suitable promoter and/or polyadenylation signal known in the art or described herein operably linked to any of the polynucleotide encoding FLT3L and/or to the polynucleotide encoding the TAP inhibitor.
  • the TAP inhibitor is derived from herpes virus 1 or herpes virus 2. In some embodiments, the TAP inhibitor is derived from bovine herpes virus 1. In some embodiments, the TAP inhibitor is any of UL49.5, US6, or ICP47. In some embodiments, the TAP inhibitor is UL49.5
  • the expression cassette further comprises a polynucleotide encoding a self-cleaving peptide.
  • Any suitable self-cleaving peptide may be used in the cassettes of the disclosure, including, but not limited to, a T2A, P2A, E2A, or F2A peptide.
  • the encoded self-cleaving peptide is a P2A peptide.
  • the encoded P2A comprises the amino acid sequence of SEQ ID NO:91In some embodiments, the self-cleaving peptide is positioned between the polynucleotide encoding the FLT3L and the polynucleotide encoding the TAP inhibitor in the expression cassette.
  • the expression cassette comprises a promoter operably linked to the polynucleotide encoding the FLT3L.
  • the promoter is the hCMV promoter.
  • the hCMV promoter comprises the nucleotide sequence of SEQ ID NO: 107
  • the expression cassette further comprises a polyadenylation signal operably linked to the polynucleotide encoding the TAP inhibitor.
  • the polyadenylation sequence is a BGHpA polyadenylation signal.
  • the BGHpA polyadenylation signal comprises the nucleotide sequence of SEQ ID NO: 102.
  • an expression cassette of the disclosure comprises, in order, a promoter (e.g., an hCMV promoter) operably linked to the polynucleotide encoding the FLT3L; the polynucleotide encoding the FLT3L; the polynucleotide encoding the self-cleaving peptide (e.g., a P2A peptide); the polynucleotide encoding the TAP inhibitor (e.g., a UL49.5 protein); and a polyadenylation signal (e.g., a BGHpA polyadenylation signal).
  • a promoter e.g., an hCMV promoter
  • an expression cassette of the disclosure comprises a polynucleotide encoding, in order, the FLT3L, the self-cleaving peptide (e.g., a P2A peptide), and the TAP inhibitor (e.g., a UL49.5 protein).
  • said polynucleotide comprises the nucleotide sequence of SEQ ID NO: 106.
  • the expression cassette encodes a polypeptide comprising, in order, the FLT3L, the self-cleaving peptide (e.g., a P2A peptide), and the TAP inhibitor (e.g., a UL49.5 protein).
  • the expression cassette further comprises a promoter, e.g., an hCMV promoter, that regulates expression of the polynucleotide encoding the FLT3L, the self-cleaving peptide (e.g., a P2A peptide), and the TAP inhibitor (e.g., a UL49.5 protein).
  • the expression cassette further comprises a polyadenylation signal, e.g., a BGHpA.
  • the expression cassette comprises, in order, a promoter, e.g., an hCMV promoter; a polynucleotide encoding, in order, the FLT3L, the self-cleaving peptide (e.g., a P2A peptide), and the TAP inhibitor (e.g., a UL49.5 protein); and a polyadenylation signal, e.g., a BGHpA.
  • a promoter e.g., an hCMV promoter
  • a polynucleotide encoding, in order, the FLT3L, the self-cleaving peptide (e.g., a P2A peptide), and the TAP inhibitor e.g., a UL49.5 protein
  • a polyadenylation signal e.g., a BGHpA.
  • an expression cassette of the disclosure comprises the nucleotide sequence of SEQ ID NO: 100, or a nucleotide sequence having any of about at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the nucleotide sequence set forth in SEQ ID NO: 100.
  • an expression cassette of the disclosure is integrated into a genome of a virus, such as an oncolytic HSV, e.g., an HSV-1 or HSV-2.
  • the cassette is integrated into one or two of the native ⁇ 34.5 loci of an oncolytic HSV, e.g., an HSV-1 or HSV- 2.
  • one or two of the native ⁇ 34.5 loci of an oncolytic HSV, e.g., an HSV-1 or HSV-2 are rendered inactive by insertion of the expression cassette.
  • integration of the expression cassette into a ⁇ 34.5 locus comprises replacing all or a part of the native ⁇ 34.5 locus with the expression cassette.
  • the TAP inhibitor encoded by the expression cassette is expressed as an immediate-early gene during viral replication.
  • the expression cassette comprises: (i) the polynucleotide encoding the TAP inhibitor (e.g., a UL49.5 protein), (ii) the polynucleotide encoding the self-cleaving peptide (such as a P2A peptide), and (iii) the polynucleotide encoding the FLT3L, e.g., as described above.
  • the expression cassette is integrated in (e.g., replaces) the native ⁇ 34.5 locus within the long terminal repeat (TRL) region of the genome, e.g. an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome.
  • the expression cassette is in the orientation relative to the unique long (UL) region of the genome, e.g. an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome, of (i)-(ii)-(iii)-UL.
  • UL unique long
  • the expression cassette comprises: (i) the polynucleotide encoding the TAP inhibitor (e.g., a UL49.5 protein), (ii) the polynucleotide encoding the self-cleaving peptide (e.g., a P2A peptide), and (iii) the polynucleotide encoding the FLT3L, e.g., as described above.
  • the expression cassette is integrated in (e.g., replaces) the native ⁇ 34.5 locus within the internal long repeat (IRL) region of the genome, e.g. an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome.
  • the expression cassette is in the orientation relative to the unique long (UL) region of the genome, e.g. an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome, of UL-(iii)-(ii)-(i).
  • UL unique long
  • V-C Oncolytic viruses, genomes, vectors and cells comprising one or more expression cassettes
  • an oncolytic virus e.g., an oncolytic HSV, such as an oncolytic HSV-1 or oncolytic HSV-2
  • the oncolytic virus comprises one or more expression cassettes comprising a polynucleotide encoding IL- 12, a polynucleotide encoding a CD40 agonist, and/or a polynucleotide encoding a CTLA-4 binding protein, e.g., as described above in Section IV-A.
  • the oncolytic virus comprises one or more expression cassettes comprising a polynucleotide encoding FLT3L and/or a polynucleotide encoding a transporter associated with antigen processing (TAP) inhibitor, e.g., as described above in Section IV-B.
  • TEP antigen processing
  • the oncolytic virus comprises: (a) one or more expression cassettes comprising a polynucleotide encoding IL- 12, a polynucleotide encoding a CD40 agonist, and/or a polynucleotide encoding a CTLA-4 binding protein, e.g., as described above in Section IV-A; and (b) one or more expression cassettes comprising a polynucleotide encoding FLT3L and/or a polynucleotide encoding a transporter associated with antigen processing (TAP) inhibitor, e.g., as described above in Section IV-B.
  • TEP antigen processing
  • an oncolytic virus of the disclosure exhibits increased T cell activation relative to an oncolytic virus lacking any one, any two, or all of the polynucleotides encoding the IL- 12 protein, the CD40 agonist, and the CTLA-4 binding protein.
  • T cell activation may be assessed using any suitable method known in the art, such as using an in vitro IL-2 secretion assay.
  • an oncolytic virus of the disclosure has increased abscopal effect relative to an oncolytic virus lacking any one, any two, or any three of the FLT3L, the IL- 12, the CD40 agonist, and the CTLA-4 binding protein.
  • an oncolytic virus of the disclosure is capable of evading an individual’s immune system.
  • an oncolytic virus of the disclosure reduces or impairs viral antigen loading onto histocompatibility complex (MHC) Class I molecules for display at the cell surface, thereby reducing adaptive immune responses to the virus.
  • MHC histocompatibility complex
  • a modified HSV genome (e.g., an HSV-1 or HSV-2 genome) comprising one or more of the expression cassettes described above (e.g., in Sections IV-A and IV-B).
  • the modified HSV genome comprises one or more expression cassettes comprising a polynucleotide encoding IL- 12, a polynucleotide encoding a CD40 agonist, and/or a polynucleotide encoding a CTLA-4 binding protein, e.g., as described above in Section IV-A.
  • the modified HSV genome comprises one or more expression cassettes comprising a polynucleotide encoding FLT3L and/or a polynucleotide encoding a transporter associated with antigen processing (TAP) inhibitor, e.g., as described above in Section IV-B.
  • TEP antigen processing
  • the modified HSV genome comprises: (a) one or more expression cassettes comprising a polynucleotide encoding IL- 12, a polynucleotide encoding a CD40 agonist, and/or a polynucleotide encoding a CTLA-4 binding protein, e.g., as described above in Section IV-A; and (b) one or more expression cassettes comprising a polynucleotide encoding FLT3L and/or a polynucleotide encoding a transporter associated with antigen processing (TAP) inhibitor, e.g., as described above in Section IV-B.
  • TEP antigen processing
  • a vector comprising one or more of the expression cassettes described above (e.g., in Sections IV-A and IV-B).
  • Suitable vectors include, without limitation, cloning vectors and expression vectors.
  • Suitable cloning vectors can be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors generally have the ability to self -replicate, may possess a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in selecting clones containing the vector.
  • Suitable examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR.322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28.
  • plasmids and bacterial viruses e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR.322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28.
  • cloning vectors are available from commercial vendors such as BioRad, Strategene, and Invitrogen.
  • Expression vectors generally are replicable polynucleotide constructs that contain a nucle
  • the expression vector may be replicable in the host cells either as episomes or as an integral part of the chromosomal DNA.
  • Suitable expression vectors include but are not limited to plasmids, viral vectors, including adenoviruses, adeno-associated viruses, HSV viruses, e.g. HSV-1 or HSV-2, retroviruses, and cosmids.
  • Vector components may generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcriptional controlling elements (such as promoters, enhancers and terminator).
  • suitable transcriptional controlling elements such as promoters, enhancers and terminator
  • one or more translational controlling elements are also usually included, such as ribosome binding sites, translation initiation sites, and stop codons.
  • cells such as host cells, comprising one or more of the expression cassettes described above (e.g., in Sections IV-A and IV-B) are also provided.
  • the cell is an isolated cell.
  • An isolated cell is a cell that is identified and separated from at least one contaminant cell with which it is ordinarily associated in the environment in which it was produced.
  • the isolated cell is free of association with all components associated with the production environment.
  • the isolated cell is in a form other than in the form or setting in which it is found in nature. Isolated cells are distinguished from cells existing naturally in tissues, organs, or individuals.
  • the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell, human cells such as HELA cells, HEK293 cells, etc., or lymphoid cell (e.g., Y0, NSO, Sp20 cell).
  • Host cells of the present disclosure also include, without limitation, isolated cells, in vitro cultured cells, and ex vivo cultured cells.
  • the cell is a mammalian cell.
  • JP-OV-2 is a next-generation recombinant HSV Type-1-based oncolytic virus (OV) that has been modified to 1) reduce innate and adaptive antiviral host responses that shut down viral replication to allow greater lysis of target tumor cells and 2) to enhance all stages of the adaptive immune response to the cancer cells through expression of multiple synergistic immune stimulatory payloads.
  • OV HSV Type-1-based oncolytic virus
  • JP-OV-2 comprises: a. a cassette integrated in one or both of the ⁇ 34.5 loci comprising in order, from upstream to downstream, a CMV promoter, a polynucleotide encoding hFLT3L, a P2A cleavage sequence, a polynucleotide encoding UL49.5, and a polyadenylation signal; and b.
  • a polynucleotide comprising in order, from upstream to downstream, a polynucleotide comprising a variant US11 gene encoding native US11 protein, an additional polynucleotide encoding native US11 protein, a polynucleotide encoding US 10 protein, a polyadenylation signal that is operably linked to the polynucleotide encoding the US 10 protein, a CMV promoter, a polynucleotide encoding a CTLA-4 binding protein, a polyadenylation signal that is operably linked to the polynucleotide encoding the CTLA-4 binding protein, a polyadenylation signal that is operably linked to a polynucleotide encoding a CD40 agonist, a polynucleotide encoding a CD40 agonist, an AoHVl promoter that controls expression of the CD40 agonist, an MMLV promoter that controls expression of an IL- 12, a
  • JP-OV-2 comprises a polynucleotide for hFLT3L encoding the amino acid sequence set forth in SEQ ID NO:71.
  • JP-OV-2 comprises a polynucleotide for UL49.5 encoding the amino acid sequence set forth in SEQ ID NO: 82.
  • JP-OV-2 comprises a polynucleotide for IL- 12 encoding the amino acid sequence set forth in SEQ ID NO: 4.
  • JP-OV-2 comprises a polynucleotide for CD40 agonist encoding the amino acid sequence set forth in SEQ ID NO: 25,
  • JP-OV-2 comprises a polynucleotide for CTLA-4 binding protein encoding the amino acid sequence set forth in SEQ ID NO: 50.
  • JP-OV-2 comprises a polynucleotide for variant US11 gene comprising the polynucleotide sequence set forth in SEQ ID NO: 204.
  • JP-OV-2 comprises an additional polynucleotide encoding for US11 encoding the amino acid sequence set forth in SEQ ID NO: 80.
  • JP-OV-2 comprises a polynucleotide for US 10 encoding the amino acid sequence set forth in SEQ ID NO:90.
  • the oncolytic viruses (such as the oncolytic HSV) described herein may be prepared using any methods known in the art or as described herein.
  • the oncolytic virus (such as the oncolytic HSV) may be engineered (such as to comprise one or more of the expression cassettes described herein and/or to express one or more of the pay load proteins described herein) by modifying a wild-type virus (such as a wild-type HSV-1) genome.
  • Transgenes and/or expression cassettes including as otherwise described herein, may be inserted in the native genome or replace native portions of the genome using recombinant cloning techniques well known in the art. Exemplary engineering methods are described herein at Examples 4-7.
  • Engineered oncolytic virus genomes may be propagated in suitable cells and collected from cell media or collected from cell lysates.
  • the virus-containing cell media or virus-containing cell lysates may then be sterilized, such as by filtration or other suitable means.
  • the virus may be concentrated, such as by ultracentrifugation.
  • Purified virus may be stored by suitable means, including by storage at about -80 °C in DMEM. Titers of virus stocks vary by orders of magnitude, depending upon the viral genotype and the protocol used to prepare and purify them. Purified virus may be titered using assays well known in the art. Viral titer may be expressed in terms of infectious viral units, such as plaque-forming units (pfu). The integrity and sequence of the viral genome may be assessed by techniques well known in the art, including whole-genome sequencing.
  • the present disclosure in some aspects provides a method of treating an advanced solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual every two weeks (Q2W) intratumorally.
  • HSV-1 virus is JP-OV-2.
  • the individual has an advanced tumor.
  • the individual has a metastatic solid tumor.
  • the individual does not have a central nervous system solid tumor.
  • the individual has previously received available standard therapy and has progressed.
  • progressive disease is defined as an increase in the sum of lesion diameters ⁇ 20% and ⁇ 5 mm from nadir.
  • the individual cannot tolerate standard therapy.
  • the individual has refused standard therapy.
  • the individual has an advanced or metastatic solid tumor for which there is no standard of care per regional guidelines.
  • the individual has at least 1 injectable tumor ⁇ 1 cm in longest diameter (or shortest diameter for lymph nodes). In some embodiments, the individual has injectable tumors that in aggregate are ⁇ 1 cm in the longest diameter. In some embodiments, the injectable tumors is measurable by RECIST v1.1 criteria. In some embodiments, the injectable lesion is not invading or in close proximity to major or large blood vessels. In some embodiments, the injectable lesion is not invading major airways.
  • the individual has at least one measurable lesion that will not be injected during the treatment.
  • the individual has a histologically or cytologically confirmed non- small cell lung cancer (NSCLC).
  • NSCLC non- small cell lung cancer
  • the individual has stage IIIB-IV NSCLC according to the 8th edition of the American Joint Committee on Cancer (AJCC) TNM staging for NSCLC.
  • the TNM staging system for NSCLC comprises T for characteristics of the primary tumor, N for nodal involvement, and M for (distant) metastasis.
  • specific T, N, and M categories that exhibit similar behavior are coalesced into stage groups.
  • the individual has stage IIIB NSCLC.
  • stage IIIB NSCLC is selected from the group consisting of T1b/N3/M0, T1c/N3/M0, T2a/N3/M0, T2b/N3/M0, T3/N2/M0, and T4/N2/M0 NSCLC.
  • the individual has stage IIIC NSCLC.
  • stage IIIC NSCLC comprises T4/N3/M0 NSCLC.
  • the individual has stage IVA NSCLC.
  • the stage IVA NSCLC comprises Tany/Nany/M1a NSCLC, or Tany/Nany/M1b NSCLC.
  • the individual has stage IVB NSCLC.
  • the stage IVB NSCLC comprises Tany/Nany/Mlc NSCLC.
  • the individual with histologically or cytologically confirmed NSCLC, stage IIIB-IV has been previously treated with an anti-PD-1/PD-L1 therapy and platinum- based chemotherapy, either as combination or sequentially for metastatic disease and has progressed on or after therapy.
  • the individual cannot tolerate or has previously refused platinum-based chemotherapy or were unable to receive platinum-based chemotherapy have progressed after anti-PD-1/PD-L1 therapy alone.
  • the stage IIIB-IV NSCLC is relapsed NSCLC.
  • the stage IIIB-IV NSCLC is refractory NSCLC.
  • the individual has an ECOG performance status of Grade 0 or 1. In some embodiments, the individual has an ECOG performance status of Grade 0. In some embodiments, the individual with an ECOG performance status of Grade 0 is fully active and able to carry on all pre-disease performance without restriction. In some embodiments, the individual has an ECOG performance status of Grade 1. In some embodiments, the individual with an ECOG performance status of Grade 1 is restricted in physically strenuous activity but ambulatory and able to carry out work of a light or sedentary nature.
  • the individual does not have active disease involvement of the central nervous system.
  • the active disease involvement of the central nervous system comprises primary central nervous system tumors, metastases, or leptomeningeal disease.
  • the individual has brain metastases that are definitively, locally treated, clinically stable and asymptomatic for more than two weeks, and who are not receiving steroids or receiving low dose corticosteroid treatment for at least 2 weeks prior to treatment with the oncolytic herpes simplex type 1 (HSV-1) virus.
  • low does corticosteroid treatment comprises less than about 10 mg prednisone or equivalent.
  • the individual does not have prior or concurrent second malignancy that due to natural history or treatment is likely to interfere with treatment with the oncolytic herpes simplex type 1 (HSV-1) virus.
  • HSV-1 oncolytic herpes simplex type 1
  • the individual does not have an active herpetic infection. In some embodiments, the individual does not have a prior history of herpetic infection. In some embodiments, the individual does not have active herpetic infections that require ongoing systemic anti-viral therapy. In some embodiments, herpetic infections comprise herpetic keratitis or encephalitis.
  • the individual does not have an active infection or condition that requires treatment with systemic anti-infective agents within about 7 days prior to the first dose of an oncolytic herpes simplex type 1 (HSV-1) virus.
  • the individual does not have an active infection or condition that requires chronic treatment with systemic anti-infective agents.
  • anti-infective agents comprise antibiotics, antifungals, or antivirals.
  • the individual does not have active autoimmune disease that requires systemic immunosuppressive medications within 12 months prior to treatment with an oncolytic herpes simplex type 1 (HSV-1) virus.
  • systemic immunosuppressive medications comprise chronic corticosteroid, methotrexate, or tacrolimus.
  • the individual is not immunocompromised. In some embodiments, the individual does not have a known positive test result for HIV or other immunodeficiency syndrome.
  • the individual does not have a history of non- infectious pneumonitis that required systemic treatment with corticosteroids. In some embodiments, the individual does not have a history of interstitial lung disease that required systemic treatment with corticosteroids. In some embodiments, the individual does not have a history of non-infectious pneumonitis that required continuous supplemental oxygen use to maintain adequate oxygenation. In some embodiments, the individual does not have a history of interstitial lung disease that required continuous supplemental oxygen use to maintain adequate oxygenation.
  • the individual does not have a history of solid organ transplantation. In some embodiments, the individual does not have a history of hematologic stem cell transplantation.
  • the individual does not have an active bleeding diathesis or requirement for therapeutic anticoagulation that cannot be interrupted or altered for procedures.
  • the individual does not have venous thromboembolic events within 1 month prior to the first dose of an oncolytic herpes simplex type 1 (HSV-1) virus.
  • the venous thromboembolic event comprises a pulmonary embolism.
  • the individual does not have clinically significant cardiovascular disease within 6 months prior to treatment with the oncolytic herpes simplex type 1 (HSV-1) virus.
  • the clinically significant cardiovascular disease is selected from the group consisting of myocardial infarction, severe or unstable angina, or coronary artery bypass surgery, clinically significant arrhythmias, congestive heart failure such as NYHA class III or V, cerebrovascular accident, transient ischemic attack, or other arterial thromboembolic event, and Myocarditis.
  • the individual has not had prior treatment with an HSV-based oncolytic virus for the treatment of metastatic disease.
  • the individual has not received prior anti-PD(L)-1 or anti-CTLA-4 therapy within 4 weeks, or other anti cancer therapy within 14 days, before the first dose of HSV treatment.
  • the individual has not received radiation therapy within 7 days before the first dose of HSV treatment.
  • the individual has not received immunosuppressive doses of systemic medication, such as corticosteroids within 7 days prior to the first dose of HSV treatment.
  • systemic medication such as corticosteroids
  • the individual has not received any live vaccine within 28 days before the first dose of HSV treatment.
  • HSV-1 herpes simplex type 1
  • the present application in one aspect provides a method of treating an advanced solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual every two weeks (Q2W) intratumorally.
  • the oncolytic virus comprises one or more expression cassettes comprising a polynucleotide encoding hFLT3L protein, a polynucleotide encoding a CTLA-4 binding protein, a polynucleotide encoding a CD40 agonist, and a polynucleotide encoding IL- 12.
  • the HSV-1 virus is JP-OV- 2.
  • the method further comprises administering an antibody that binds to PD-1 every four weeks (Q4W).
  • the antibody that binds to PD-1 is administered intravenously.
  • the antibody that binds to PD-1 is administered to the individual at a dose of about 480 mg.
  • cetrelimab In some embodiments, about 480 mg of cetrelimab is administered Q4W intravenously to the individual.
  • the present application in one aspect provides a method of treating an advanced solid tumor in an individual comprising administering an oncolytic HSV-1 virus to the individual intratumorally Q2W and administering an antibody that binds to PD-1 to the individual intravenously Q4W, wherein the first administration of the HSV-1 virus occurs on week
  • the first administration of the PD-1 antibody occurs on week 3.
  • the first administration of the PD-1 antibody occurs on week 3.
  • HSV-1 virus is JP-OV-2. In some embodiments, the HSV-1 virus is administered at a dose of about
  • the HSV-1 virus is administered at a dose of about 10 5 PFU/mL. In some embodiments, the HSV-1 virus is administered at a dose of about 10 6 PFU/mL. In some embodiments, the HSV-1 virus is administered at a dose of about 10 7 PFU/mL. In some embodiments, the HSV-1 virus is administered at a dose of about 10 8 PFU/mL.
  • the antibody that binds to PD-1 is cetrelimab. In some embodiments, about 480 mg of cetrelimab is administered intravenously to the individual during each administration. In some embodiments, about 480 mg of cetrelimab is administered intravenously to the individual during each administration and after administration of the HSV-1 virus.
  • a method of treating an advanced or metastatic non-central nervous system tumor in an individual comprising administering about 10 5 PFU/mL, about 10 6 PFU/mL, about 10 7 PFU/mL, or about 10 8 PFU/mL of an HSV-1 virus and administering an antibody that binds to PD-1 to the individual.
  • the HSV-1 virus is administered at a dose of about 10 5 PFU/mL.
  • the HSV-1 virus is administered at a dose of about 10 6 PFU/mL.
  • the HSV-1 virus is administered at a dose of about 10 7 PFU/mL.
  • the HSV-1 virus is administered at a dose of about 10 8 PFU/mL. In some embodiments, the individual has received all available standard therapies and progressed. In some embodiments, the HSV-1 virus is JP-OV- 2. In some embodiments, the HSV-1 virus is administered via intratumoral injection. In some embodiments, the individual is administered the HSV-1 virus via a superficial approach comprising injection under direct visualization and palpation into a cutaneous, subcutaneous, or nodal lesion. In some embodiments, the individual is administered the HSV-1 virus via a percutaneous approach for lesions accessible via imaging guidance. In some embodiments, the percutaneous administration approach is used for transthoracically accessible lung lesions, transabdominally accessible liver lesions, or subcutaneous soft tissue lesions.
  • the HSV-1 virus is administered to the individual intratumorally Q2W between about 1 to about 8 times. In some embodiments, the HSV-1 virus is administered to the individual 1, 2, 3, 4, 5, 6, 7, or 8 times. In some embodiments, the HSV-1 virus is JP-OV- 2.
  • the individual receives intravenous administration of an antibody that binds to PD-1 Q4W for up to a total of about two years.
  • the antibody that binds to PD-1 is cetrelimab.
  • a method of treating an advanced solid tumor in an individual comprising administering an oncolytic HSV-1 virus to the individual intratumorally Q2W and administering an antibody that binds to PD-1 to the individual intravenously Q4W, wherein the first administration of the HSV-1 virus occurs on week 1 and the first administration of the PD-1 antibody occurs on week 3.
  • the HSV-1 virus is JP-OV-2.
  • the antibody that binds to PD-1 is cetrelimab.
  • the individual has an advanced or metastatic non-central nervous system tumor. In some embodiments, the individual has received all available standard therapies and progressed.
  • the method of treating a solid tumor in an individual comprises i) administering an HSV-1 virus intratumorally Q2W on weeks 1, 3, 5, 7, 9, 11, 13, and 15, and ii) administering an antibody that binds to PD-1 intravenously Q4W on weeks 3, 7, 11, and 15.
  • the present application in one aspect provides a method of treating advanced metastatic NSCLC in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual intratumorally Q2W and administering an antibody that binds to PD-1 to the individual intravenously Q4W, wherein the first administration of the HSV-1 virus and the first administration of the PD-1 antibody occurs on week 1.
  • HSV-1 virus is administered at a dose of about 10 5 PFU/mL, about 10 6 PFU/mL, about 10 7 PFU/mL, or about 10 8 PFU/mL.
  • the HSV-1 virus is administered at a dose of about 10 5 PFU/mL.
  • the HSV-1 virus is administered at a dose of about 10 6 PFU/mL. In some embodiments, the HSV-1 virus is administered at a dose of about 10 7 PFU/m. In some embodiments, the HSV-1 virus is administered at a dose of about 10 8 PFU/mL. In some embodiments the HSV-1 virus is JP-OV-2.
  • the antibody that binds to PD-1 is cetrelimab. In some embodiments, about 480 mg of cetrelimab is administered intravenously to the individual during each administration. In some embodiments, about 480 mg of cetrelimab is administered intravenously to the individual during each administration and after administration of the HSV-1 virus.
  • a method of treating advanced metastatic NSCLC in an individual comprising administering about 10 5 PFU/mL, about 10 6 PFU/mL, about 10 7 PFU/mL, or about 10 8 PFU/mL of an HSV-1 virus and administering an antibody that binds to PD-1 to the individual.
  • the HSV-1 virus is administered at a dose of about 10 5 PFU/mL.
  • the HSV-1 virus is administered at a dose of about 10 6 PFU/mL.
  • the HSV-1 virus is administered at a dose of about 10 7 PFU/m.
  • the HSV-1 virus is administered at a dose of about 10 8 PFU/mL. In some embodiments the HSV-1 virus is JP-OV-2. In some embodiments, the individual has progressed after treatment with an anti-PD-1 antibody and chemotherapy. In some embodiments, the individual has stage IIIB-IV NSCLC according to the 8th edition of the American Joint Committee on Cancer (AJCC) TNM staging for NSCLC. In some embodiments, the individual has at least 2 measurable lesions, a first measurable lesion that is accessible for endobronchial or percutaneous injection, and a second measurable lesion that will not be injected. In some embodiments, the individual has a maximum of 5 measurable lesions in total at the start of treatment. In some embodiments, the individual does not have more than 2 measurable lesions per organ at the start of treatment. In some embodiments, the HSV-1 virus is administered intratumorally Q2W. In some embodiments the PD-1 antibody is administered intravenously Q4W.
  • the HSV-1 virus is administered to an individual with NSCLC via intratumoral injection.
  • the individual is administered the HSV-1 virus via a superficial approach with injection under direct visualization and palpation into a cutaneous, subcutaneous, or nodal lesion.
  • the individual is administered the HSV-1 virus via a percutaneous approach for lesions accessible via imaging guidance.
  • the percutaneous administration approach is selected for transthoracically accessible lung lesions, transabdominally accessible liver lesions, or subcutaneous soft tissue lesions.
  • the individual is administered the HSV-1 virus via an endobronchial approach for lung lesions and lymph nodes accessible via a bronchoscope.
  • the HSV-1 virus is JP-OV-2.
  • the HSV-1 virus is administered to an individual with NSCLC intratumorally Q2W between about 1 to about 8 times. In some embodiments, the HSV-1 virus is administered to the individual 1, 2, 3, 4, 5, 6, 7, or 8 times. In some embodiments, the HSV-1 virus is administered to the individual 8 times. In some embodiments, the individual receives at most 8 treatments with the HSV-1 virus. In some embodiments, the individual is administered the HSV- 1 virus intratumorally Q2W for between about 2 and about 16 weeks. In some embodiments, the individual is administered the HSV-1 virus intratumorally Q2W for about 2 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 14 weeks or about 16 weeks. In some embodiments, the individual is administered the HSV-1 virus intratumorally Q2W for about 16 weeks. In some embodiments the HSV-1 virus is JP-OV-2.
  • the individual receives intravenous administration of an antibody that binds to PD-1 Q4W for up to a total of about two years.
  • the antibody that binds to PD-1 is cetrelimab.
  • the present application in one aspect provides a method of treating advanced metastatic NSCLC in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual intratumorally Q2W and administering an antibody that binds to PD-1 to the individual intravenously Q4W, wherein the first administration of the HSV-1 virus and the first administration of the PD-1 antibody occurs on week 1.
  • HSV-1 virus is JP-OV-2.
  • the antibody that binds to PF-1 is cetrelimab.
  • the individual has progressed after treatment with an anti- PD-1 antibody and chemotherapy.
  • the individual has stage IIIB-IV NSCLC according to the 8th edition of the American Joint Committee on Cancer (AJCC) TNM staging for NSCLC.
  • the method of treating advanced metastatic NSCLC in an individual comprises i) administering an HSV-1 virus intratumorally Q2W on weeks 1, 3, 5, 7, 9, 11, 13, and 15, and ii) administering an antibody that binds to PD-1 intravenously Q4W on weeks 1, 5, 9, 13, and 15.
  • the HSV-1 virus is administered in a maximum volume of aboutlO ml. In some embodiments, the HSV-1 virus is administered in an injection volume calculated based on lesion size. In some embodiments, the HSV-1 virus is administered to a superficial lesion or a visceral lesion. In some embodiments, the HSV-1 virus is administered in a volume of about 4 ml for a superficial lesion comprising a lesion size greater than at least 5 cm in the longest dimension. In some embodiments, the HSV-1 virus is administered in a volume of about 3 ml for a superficial lesion comprising a lesion size in the longest dimension of between about 4 cm and about 5 cm.
  • the HSV-1 virus is administered in a volume of about 2 ml for a superficial lesion comprising a lesion size in the longest dimension of between about 3 cm and about 4 cm. In some embodiments, the HSV-1 virus is administered in a volume of about 1.5 ml for a superficial lesion comprising a lesion size in the longest dimension of between about 2 cm and about 3 cm. In some embodiments, the HSV-1 virus is administered in a volume of about 0.5 ml for a superficial lesion comprising a lesion size in the longest dimension of between about 1.5 cm and about 2 cm.
  • the HSV-1 virus is administered in a volume of about 0.3 ml for a superficial lesion comprising a lesion size in the longest dimension of between about 1 cm and about 1.5 cm. In some embodiments, the HSV-1 virus is administered in a volume of about 0.2 ml for a superficial lesion comprising a lesion size in the longest dimension of less than about 1 cm. In some embodiments, large superficial lesions may be injected using a corresponding visceral injection volume for the size of the given superficial lesion. In some embodiments, the full amount of HSV-1 virus cannot be administered to the lesion due to changes in lesion size, for example a lesion may decrease in size following a previous disease assessment. In some embodiments, the pressure required to administer the full amount of HSV-1 virus to the lesion is prohibitive in the opinion of the treating physician. In some embodiments the HSV-1 virus is JP- OV-2.
  • the HSV-1 virus is administered in a maximum volume of about 10 ml. In some embodiments, the HSV-1 virus is administered in an injection volume calculated based on lesion size. In some embodiments, the HSV-1 virus is administered to a superficial lesion or a visceral lesion. In some embodiments, the HSV-1 virus is administered in a volume of about 10 ml for a visceral lesion comprising a lesion size greater than at least 5 cm in the longest dimension. In some embodiments, the HSV-1 virus is administered in a volume of about 8 ml for a visceral lesion comprising a lesion size in the longest dimension of between about 4 cm and about 5 cm.
  • the HSV-1 virus is administered in a volume of about 6 ml for a visceral lesion comprising a lesion size in the longest dimension of between about 3.5 cm and about 4 cm. In some embodiments, the HSV-1 virus is administered in a volume of about 4 ml for a visceral lesion comprising a lesion size in the longest dimension of between about 2.5 cm and about 3.5 cm. In some embodiments, the HSV-1 virus is administered in a volume of about 2 ml for a visceral lesion comprising a lesion size in the longest dimension of between about 2 cm and about 2.5 cm.
  • the HSV-1 virus is administered in a volume of about 1 ml for a visceral lesion comprising a lesion size in the longest dimension of between about 1.5 cm and about 2 cm. In some embodiments, the HSV-1 virus is administered in a volume of about 0.5 ml for a visceral lesion comprising a lesion size in the longest dimension of between about 1 cm and about 1.5 cm. In some embodiments, the full amount of HSV-1 virus cannot be administered to the lesion due to changes in lesion size, for example a lesion may decrease in size following a previous disease assessment. In some embodiments, the pressure required to administer the full amount of HSV-1 virus to the lesion is prohibitive in the opinion of the treating physician. In some embodiments the HSV-1 virus is JP-OV-2.
  • one or more lesions are injected with HSV-1 virus during each administration. In some embodiments, regardless of the number of one or more lesions injected, the maximum total HSV-1 dose volume injected across all lesions is no more than about 10 mL. In some embodiments the HSV-1 virus is JP-OV-2.
  • lesions are selected for injection in an individual with NSCLC. In some embodiments, only lesions in one hemithorax can be injected per administration. In some embodiments, injections of the HSV-1 virus into the lung may involve endobronchial injection via a bronchoscope. In some embodiments, injections of the HSV-1 virus into the lung may involve percutaneous image-guided injections for lesions that are not accessible by a bronchoscope. In some embodiments, at least 1 lesion is selected as an uninjectable target lesion unless all other lesions become uninjectable. In some embodiments, during the first administration of HSV-1 virus the largest injectable lesion is selected as a target lesion for injection of the HSV-1 virus. In some embodiments, the largest injectable lung lesion is prioritized for injection. In some embodiments, primary and affected measurable lymph nodes are selected for injection subsequent to the largest injectable lung lesion.
  • the HSV-1 virus is injected into a superficial lesion.
  • a superficial lesion comprises a cutaneous, subcutaneous, or nodal lesion that can be accessed in the clinic under direct visualization/palpation.
  • the HSV-1 virus is injected into a cutaneous, subcutaneous, or nodal lesion.
  • the HSV- 1 virus is JP-OV-2.
  • the HSV-1 virus is injected into a visceral lesion or a deep nodal lesion.
  • the HSV-1 virus is JP-OV-2.
  • injecting the HSV-1 virus into a visceral lesion requires imaging guidance, for example ultrasound, CBCT, or CT imaging.
  • the HSV-1 virus is not injected into visceral lesions with major airway or blood vessel invasion.
  • the HSV-1 virus is not injected into visceral lesions with a history of poor wound healing. In some embodiments, no more than 3 visceral lesions within a single solid organ should be injected at a single administration.
  • the HSV-1 virus is injected into a transthoracically accessible lung lesion, a transabdominally accessible liver lesion, and/or a subcutaneous soft tissue lesion.
  • the HSV-1 virus is administered to a lung lesion or a lymph node accessible via a bronchoscope.
  • the HSV-1 virus is JP-OV-2.
  • the present application provides a method of treating an advanced solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual every two weeks (Q2W) intratumorally in combination with administration of an antibody that binds to PD-1 every four weeks (Q4W).
  • HSV-1 virus is JP-OV-2.
  • the antibody that binds to PD-1 is administered intravenously.
  • the antibody that binds to PD-1 is administered to the individual at a dose of about 480 mg.
  • the antibody that binds to PD-1 is cetrelimab.
  • about 480 mg of cetrelimab is administered intravenously to the individual about every 4 weeks (Q4W) for at least about 15 weeks.
  • the antibody that specifically binds to PD-1 comprises a heavy chain and a light chain
  • the heavy chain of the antibody comprises a heavy chain variable region (VH) comprising a CDR-H1, CDR-H2, and CDR-H3 of a VH comprising the amino acid sequence set forth in SEQ ID NO:400
  • the light chain of the antibody comprises a light chain variable region (VL) comprising a CDR-L1, CDR-L2, and CDR-L3 of a VL comprising the amino acid sequence set forth in SEQ ID NO:404.
  • the antibody that specifically binds to PD-1 comprises a VH comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 401, a CDR- H2 comprising the amino acid sequence set forth in SEQ ID NO: 402, a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 403; and/or a VL comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 405, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 406, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 407.
  • the antibody that binds to PD-1 is administered to the individual every four weeks (Q4W). In some embodiments, the antibody that binds to PD-1 is administered to the individual every four weeks (Q4W) and after administration of the HSV-1 virus.
  • a method of treating a solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual about every two weeks (Q2W) intratumorally and administering an antibody that binds to PD-1 about every four weeks (Q4W) intravenously, for at least about 15 weeks.
  • HSV-1 virus is JP-OV-2.
  • the antibody that binds to PD-1 is cetrelimab.
  • a method of treating a solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual about every two weeks (Q2W) intratumorally for at least about 15 weeks and administering an antibody that binds to PD-1 about every four weeks (Q4W) intravenously, for at most 2 years.
  • HSV-1 virus is JP-OV-2.
  • the antibody that binds to PD- 1 is cetrelimab.
  • treatment with JP-OV-2 is initiated and continued in combination with cetrelimab, such that participants receive combination treatment starting with the first dose.
  • up to 8 treatments of JP-OV-2 may be administered, with the possibility of administering an additional 8 doses, provided that defined safety criteria are met.
  • higher ratios of injected JP-OV-2 will be achieved by limiting the largest lesion diameter for injection to 4 cm or less in order to administer a full 10 mL target JP- OV-2 dose.
  • the alternative injection volumes per lesion size are specified in Table 19, and these volumes may be evaluated on a Q2W schedule or through alternative dosing regimens.
  • a method of treating an advanced solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual every two weeks (Q2W) intratumorally, wherein the individual experiences tumor size reduction.
  • HSV-1 is JP-OV-2.
  • the method further comprises administering an antibody that binds to PD-1 about every four weeks (Q4W) intravenously.
  • the antibody that binds to PD-1 is cetrelimab.
  • the individual has more than one solid tumor.
  • the HSV-1 virus is administered to at least one of the solid tumors.
  • change in tumor size is defined as the difference in percentage change in sum of diameters of injected lesions and uninjected lesions, as measured based on RECIST vl.1. In some embodiments, such treatment results in a reduction in the Sum of Diameter (SOD) in an individual. In some embodiments, the Sum of diameter (SOD) is defined as the sum of diameter of all lesions with measurement(s).
  • the individual experiences an abscopal effect.
  • the size of a solid tumor other than the at least one solid tumor where the HSV-1 is administered decreases in size following treatment.
  • a method of treating an advanced solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual every two weeks (Q2W) intratumorally and administering an antibody that binds to PD- 1 about every four weeks (Q4W) intravenously, wherein the individual experiences a complete response.
  • an individual receiving such treatment experiences a partial response.
  • an individual receiving such treatment experiences stable disease.
  • the HSV-1 is JP-OV-2.
  • the antibody that binds to PD-1 is cetrelimab.
  • a method of treating an advanced solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual every two weeks (Q2W) intratumorally and administering an antibody that binds to PD- 1 about every four weeks (Q4W) intravenously, wherein such treatment results in an objective response rate (ORR) in a population of patients who have received such treatment.
  • HSV-1 is JP-OV-2.
  • the antibody that binds to PD-1 is cetrelimab.
  • the Objective Response Rate (ORR) is defined as the proportion of participants who have best response of Complete Response (CR) or Partial Response (PR) according to RECIST v1.1.
  • a method of treating an advanced solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual every two weeks (Q2W) intratumorally and administering an antibody that binds to PD- 1 about every four weeks (Q4W) intravenously, wherein such treatment results in a Disease Control Rate (DCR) in a population of patients who have received such treatment.
  • HSV-1 is JP-OV-2.
  • the antibody that binds to PD-1 is cetrelimab.
  • the Disease Control Rate (DCR) is defined as the percentage of participants who have achieved complete response, partial response, and stable disease.
  • a method of treating an advanced solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual every two weeks (Q2W) intratumorally and administering an antibody that binds to PD- 1 about every four weeks (Q4W) intravenously, wherein such treatment results in Duration of Response (DOR) in a population of patients who have received such treatment thereby demonstrating the subject is treated.
  • HSV-1 is JP-OV-2.
  • the antibody that binds to PD-1 is cetrelimab.
  • the Duration of Response will be calculated among responders from the date of initial documentation of a response to the date of first documented evidence of relapse according to RECIST v1.1 , or death due to any cause, whichever occurs first.
  • the DOR in individuals with disease that have not relapsed and who are alive will be censored at the last disease evaluation before the start of any subsequent anticancer therapy. In some embodiments, such treatment increases the DOR in the individual.
  • a method of treating an advanced solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual every two weeks (Q2W) intratumorally and administering an antibody that binds to PD- 1 about every four weeks (Q4W) intravenously, wherein such treatment results in a Progression Free Survival (PFS) in a population of patients who have received such treatment.
  • HSV-1 is JP-OV-2.
  • the antibody that binds to PD-1 is cetrelimab.
  • the Progression Free Survival (PFS) is defined as the time from treatment initiation until disease progression or worsening or death due to any cause. In some embodiments, such treatment increases the PFS in the individual.
  • a method of treating an advanced solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual every two weeks (Q2W) intratumorally and administering an antibody that binds to PD- 1 about every four weeks (Q4W) intravenously, wherein such treatment results in an Overall Survival (OS) in a population of patients who have received such treatment.
  • HSV-1 is JP-OV-2.
  • the antibody that binds to PD-1 is cetrelimab.
  • the Overall Survival (OS) is defined as the time from treatment initiation until death due to any cause.
  • a method of treating an advanced solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual every two weeks (Q2W) intratumorally and administering an antibody that binds to PD- 1 about every four weeks (Q4W) intravenously, wherein such treatment is safe and well-tolerated.
  • HSV-1 is JP-OV-2.
  • the antibody that binds to PD-1 is cetrelimab.
  • a method of administering a safe and tolerable treatment for an advanced solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual every two weeks (Q2W) intratumorally and administering an antibody that binds to PD-1 about every four weeks (Q4W) intravenously.
  • HSV-1 is JP-OV-2.
  • the antibody that binds to PD-1 is cetrelimab.
  • compositions comprising any of the oncolytic viruses (such as any of the oncolytic HSV) and PD-1 antibodies described herein, and optionally a pharmaceutically acceptable excipient, carrier, and/or stabilizer.
  • Pharmaceutical compositions can be prepared by mixing a oncolytic virus (such as any of the oncolytic HSV) described herein having a desired degree of purity with pharmaceutically acceptable carriers, excipients, and/or stabilizers.
  • the pharmaceutical composition comprises a pharmaceutically acceptable carrier, excipient, and/or stabilizer.
  • Pharmaceutically acceptable includes any carrier, excipient, and/or stabilizer which does not interfere with the desired effectiveness or biological activity of the oncolytic virus (such as an oncolytic HSV) and/or that is not toxic to the subject to whom the pharmaceutical composition is, or is to be, administered.
  • Pharmaceutically acceptable carriers, excipients, and stabilizers are known in the art, including in the Handbook of Pharmaceutical Excipients. Non-limiting examples of suitable carriers, excipients, and stabilizers include buffering agents preservatives, binders, compaction agents, lubricants, chelators, dispersion enhancers, disintegration agents, coloring agents, and the like.
  • kits and articles of manufacture comprising any of the oncolytic viruses (such as any of the oncolytic HSV) and PD-1 antibodies described herein, pharmaceutical compositions comprising any of the oncolytic viruses (such as any of the oncolytic HSV) and PD- 1 antibodies described herein, or isolated nucleic acids (such as the expression cassettes) described herein.
  • kits of the present application are in suitable packaging.
  • suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Kits may optionally provide additional components such as buffers and interpretative information.
  • the present application also provides articles of manufacture, which include vials, bottles, jars, flexible packaging, and the like.
  • the article of manufacture can comprise a container and a label or package insert on or associated with the container.
  • kits or article of manufacture may include multiple unit doses of the pharmaceutical composition , packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies.
  • Embodiment 1 is a method of treating an advanced solid tumor in an individual comprising administering to the individual an oncolytic herpes simplex type 1 (HSV-1) virus every two weeks (Q2W) intratumorally, wherein the HSV-1 comprises one or more expression cassettes comprising a polynucleotide encoding hFLT3L protein, a polynucleotide encoding a CTLA-4 binding protein, a polynucleotide encoding a CD40 agonist, and a polynucleotide encoding IL- 12.
  • HSV-1 oncolytic herpes simplex type 1
  • the HSV-1 comprises one or more expression cassettes comprising a polynucleotide encoding hFLT3L protein, a polynucleotide encoding a CTLA-4 binding protein, a polynucleotide encoding a CD40 agonist, and a polynucleotide encoding IL- 12.
  • Embodiment 2 is the method of embodiment 1, wherein the oncolytic HSV-1 virus comprises: a. a cassette integrated in one or both of the ⁇ 34.5 loci comprising in order, from upstream to downstream, a CMV promoter, a polynucleotide encoding hFLT3L, a P2A cleavage sequence, a polynucleotide encoding UL49.5, and a polyadenylation signal; and b.
  • a polynucleotide comprising in order, from upstream to downstream, a polynucleotide comprising a variant US11 gene encoding native US11 protein, an additional polynucleotide encoding native US11 protein, a polynucleotide encoding US 10 protein, a polyadenylation signal that is operably linked to the polynucleotide encoding the US 10 protein, a CMV promoter, a polynucleotide encoding a CTLA-4 binding protein, a polyadenylation signal that is operably linked to the polynucleotide encoding the CTLA-4 binding protein, a polyadenylation signal that is operably linked to a polynucleotide encoding a CD40 agonist, a polynucleotide encoding a CD40 agonist, an AoHVl promoter that controls expression of the CD40 agonist, an MMLV promoter that controls expression of an IL- 12, a
  • Embodiment 3 is the method of embodiment 1 or embodiment 2, wherein the oncolytic HSV-1 virus is JP-OV-2.
  • Embodiment 4 is the method of any one of embodiments 1-3, wherein the HSV-1 virus is administered to the individual as a monotherapy to treat the solid tumor.
  • Embodiment 5 is the method of any one of embodiments 1-4, wherein the HSV-1 virus is administered to the individual intratumorally in one or more lesions.
  • Embodiment 6 is the method of any one of embodiments 1-5, further comprising administering to the individual an antibody that binds to PD-1.
  • Embodiment 7 is the method of any of embodiments 1-6, wherein the individual does not have a central nervous system solid tumor.
  • Embodiment 8 is the method of any one of embodiments 1-7, wherein the individual has non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • Embodiment 9 is the method of any one of embodiments 1-8, wherein the individual has relapsed or refractory metastatic NSCLC.
  • Embodiment 10 is the method of any one of embodiments 1-9, wherein the individual has received all available standard therapy and a solid tumor progressed.
  • Embodiment 11 is the method of any one of embodiments 1-10, wherein the individual has stage IIIB-IV NSCLC.
  • Embodiment 12 is the method of any one of embodiments 1-11, wherein the individual was previously treated with (a) an anti-PD-1 or an anti-PD-L1 therapy; and (b) a platinum-based chemotherapy, either as combination or sequentially for a metastatic disease and the metastatic disease has progressed on or after therapy.
  • Embodiment 13 is the method of any one of embodiments 1-12, wherein the individual cannot tolerate or has previously refused the platinum based chemotherapy.
  • Embodiment 14 is the method of any one of embodiments 1-13, wherein the individual is unable to receive platinum-based chemotherapy and has progressed after the anti-PD-1 or the anti-PD-L1 therapy alone.
  • Embodiment 15 is the method of any one of embodiments 1-14 wherein the individual has a PD-L1 expression of less than 1% and has previously been treated with an anti-PD-1 therapy.
  • Embodiment 16 is the method of any one of embodiments 1-15 wherein the individual has a PD-L1 expression of greater than 50% and has not received a prior systemic therapy for the metastatic disease.
  • Embodiment 17 is the method of any one of embodiments 1-16, wherein the HSV-1 virus is administered to the individual at a dose of about 10 5 about, 10 6 , about 10 7 , or about 10 8 PFU/mL.
  • Embodiment 18 is the method of any one of embodiments 1-17, wherein the HSV-1 virus is administered to the individual at a dose of about 10 5 PFU/mL.
  • Embodiment 19 is the method of any one of embodiments 1-18, wherein the HSV-1 virus is administered to the individual at a dose of about 10 6 PFU/mL.
  • Embodiment 20 is the method of any one of embodiments 1-19, wherein the HSV-1 virus is administered to the individual at a dose of about 10 7 PFU/mL.
  • Embodiment 21 is the method of any one of embodiments 1-20, wherein the HSV-1 virus is administered to the individual at a dose of about 10 8 PFU/mL.
  • Embodiment 22 is the method of any one of embodiments 1-21, wherein the volume of the HSV-1 injected per lesion is determined based on the longest diameter of the lesion.
  • Embodiment 23 is the method of any one of embodiments 1-22, wherein the HSV-1 virus is administered to the individual in a maximum volume of 10 ml.
  • Embodiment 24 is the method of any one of embodiments 1-23, wherein the HSV-1 virus is injected to the individual into a cutaneous, subcutaneous, or nodal lesion.
  • Embodiment 25 is the method of embodiment 24, wherein the HSV-1 virus is administered to the cutaneous, subcutaneous, or nodal lesion in a superficial approach with injection under direct visualization and palpation.
  • Embodiment 26 is the method of any one of embodiments 1-25, wherein the HSV-1 virus is injected to the individual into a transthoracically accessible lung lesion, a transabdominally accessible liver lesion, and/or a subcutaneous soft tissue lesion.
  • Embodiment 27 is the method of embodiment 26, wherein the HSV-1 virus is administered to the transthoracically accessible lung lesion, the transabdominally accessible liver lesion, and/or the subcutaneous soft tissue lesion in a percutaneous administration approach via imaging guidance.
  • Embodiment 28 is the method of any one of embodiments 1-27, wherein the HSV-1 virus is administered to a lung lesion or a lymph node accessible via a bronchoscope.
  • Embodiment 29 is the method of any one of embodiments 1-28, wherein the individual receives a maximum of 8 doses of the HSV-1 virus.
  • Embodiment 30 is the method of any one of embodiments 6-29, wherein the antibody that specifically binds to PD-1 comprises a heavy chain variable region (VH) comprising a heavy chain complementarity-determining region (CDRH1) comprising the amino acid sequence set forth in SEQ ID NO: 401, a CDRH2 comprising the amino acid sequence set forth in SEQ ID NO: 402, a CDRH3 comprising the amino acid sequence set forth in SEQ ID NO: 403; and a light chain variable region (VL) comprising a light chain complementarity-determining region (CDRL1) comprising the amino acid sequence set forth in SEQ ID NO: 405, a CDRL2 comprising the amino acid sequence set forth in SEQ ID NO: 406, and a CDRL3 comprising the amino acid sequence set forth in SEQ ID NO: 407.
  • VH heavy chain variable region
  • CDRH1 heavy chain complementarity-determining region
  • CDRL1 light chain complementarity-determining region
  • CDRL1 light chain complementarity-determining region
  • Embodiment 31 is the method of embodiment 30, wherein the antibody that specifically binds to PD-1 comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 408 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 409.
  • Embodiment 32 is the method of embodiment 31, wherein the antibody that specifically binds to PD-1 comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 400 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 404.
  • Embodiment 33 is the method of any one of embodiments 6-32, wherein the antibody that binds to PD-1 is cetrelimab.
  • Embodiment 34 is the method of any one of embodiments 6-33, wherein the antibody that binds to PD-1 is administered to the individual at a dose of about 480 mg.
  • Embodiment 35 is the method of any one of embodiments 6-34, wherein the antibody that binds to PD-1 is administered to the individual every four weeks (Q4W).
  • Embodiment 36 is the method of any one of embodiments 6-35 wherein the antibody that binds to PD-1 is administered to the individual every four weeks (Q4W) for at least about 15 weeks.
  • Embodiment 37 is the method of embodiment 36, wherein the antibody that binds to PD- 1 is administered to the individual four weeks (Q4W) for at most about 2 years.
  • Embodiment 38 is the method of any one of embodiments 6-37, wherein the antibody that binds to PD-1 is administered intravenously.
  • Embodiment 39 is the method of any one of embodiments 6-38, wherein the antibody that binds to PD-1 is administered to the individual after the HSV-1 virus.
  • Embodiment 40 is the method of any one of embodiments 6-39, wherein the antibody that binds to PD-1 is administered to the individual starting in week three of treatment, wherein week one of treatment is the first administration of the HSV-1 virus.
  • Embodiment 41 is the method of any one of embodiments 6-40, wherein the antibody that binds to PD-1 is administered to the individual starting in week one of treatment, wherein week one of treatment is the first administration of the HSV-1 virus.
  • Embodiment 42 is the method of any one of embodiments 1-41, wherein the size of the solid tumor is reduced.
  • Embodiment 43 is the method of any one of embodiments 1-42, wherein the individual has more than one solid tumor.
  • Embodiment 44 is the method of embodiment 43, wherein the HSV-1 is administered to at least one of the solid tumors. [0285] Embodiment 44 is the method of embodiment 44, wherein the size of a solid tumor other than the at least one solid tumor where the HSV-1 is administered decreases in size following treatment.
  • Emdodiment 46 is a method of optimizing a dosing regimen of JP-OV-2 for treating an advanced solid tumor in an individual, comprising: a. evaluating the first three dose escalation levels of JP-OV-2 in a bi-weekly (Q2W) schedule; and b. implementing one or more alternative dosing regimens for JP-OV-2; wherein said alternative dosing regimens involve a dose intensification.
  • Q2W bi-weekly
  • Embodiment 47 is the method of embodiment 46, wherein the individual receives JP-OV- 2 in combination with cetrelimab starting from the first dose of JP-OV-2.
  • Embodiment 48 the method of embodiment 46 or 47, wherein the dose intensification comprises: a. administering JP-OV-2 on Days 1 and 8 of a 28-day cycle of cetrelimab as a first intensified dose; b. evaluating the safety and efficacy of the first intensified dose; and c. administering a second intensified dose of JP-OV-2 based on the evaluation of (b).
  • Embodiment 49 is the method of any one of embodiments 46-48, further comprising administering up to an additional 8 doses of JP-OV-2 after the individual has received 8 doses from an initial dosing regimen.
  • Table 7 CTLA-4 Antagonist Pay load Amino Acid Sequences.
  • EXAMPLE 1 ONCOLYTIC HSV-1 VIRUS IN COMBINATION WITH AN ANTI-PD1 ANTIBODY IN AN MC 38 5 AG TUMOR MODEL.
  • This example describes an oncolytic HSV-1 virus in combination with an anti-PD-1 antibody.
  • the genome architecture of the mouse surrogate HSV-1 virus, mJP-OV-2, and the human HSV-1 virus, JP-OV-2, are provided in FIGs. 1A-1B.
  • the mJP-OV-2 virus was engineered to express several immunomodulatory payloads including an anti-CTLA-4 antagonist (m ⁇ CTLA-4), a CD40 agonist (mCD40ag), IL- 12 (mscIL-12), and hFLT3L.
  • the virus was also engineered to express and UL49.5, codon-optimized US 11 (hCoUS11) using the US 12 immediate early promoter, and endogenous US 11 using late US 11 promoter.
  • the mouse surrogate HSV-1 virus mJP-OV-2 was engineered with 4 immunomodulatory payloads: hFLT3L, mCD40 ag, ⁇ mCTLA4, and mscIL12.8 known to activate all stages of adaptive immunity.
  • hFLT3L was retained in mJP-OV-2, as this payload is cross-reactive between mouse and human.
  • FIG. 2A depicts a diagram of the experimental workflow to assess treatment with an oncolytic HSV-1 virus in combination with an anti-PD-1 antibody.
  • the general experimental design was to implant tumor cells bilaterally, followed by intratumoral treatment of established tumors on the right flank. Treated and untreated tumors were monitored to assess if the treatment inhibited growth of the primary tumor as well as an abscopal effect on growth of the secondary untreated tumor, which would indicate that the mice had developed an adaptive immune response to tumor antigens.
  • mice Female C57BL/6 mice were obtained from Charles River Laboratories and were enrolled when they were approximately 8 weeks of age with an average body weight of 18 to 20 g.
  • the mouse syngeneic cancer cell line, MC-38-5AG was obtained from Janssen R&D, Spring House, PA, and was grown in complete culture medium. MC-38-5AG cells were harvested during exponential growth on Day 0, using TrypLE. Cells were washed twice in cold DPBS and resuspended in cold DPBS at a concentration of 5 ⁇ 10 6 cells/mL. Mice were implanted bilaterally in each flank by SC injection with 0.1 mL of the cell suspension (ie, 5 ⁇ 10 5 cells in each flank).
  • Day 0 was the day of tumor cell implantation and study initiation.
  • Cells were implanted SC in both the left and right flanks, just below the ribcage.
  • Mice were randomized by the Multi Task method in Studylog software (Studylog Systems, Version 4.3) according to tumor volume (TV), using the right side as the primary tumor, followed by the left side as the secondary tumor, such that the p value was as close to 1 as possible and the percent difference and standard deviations were similar among the groups within each cohort. All intratumoral injections were in 0.05 mL and were performed on the right tumor.
  • the vehicle control group was dosed with BeneVir formulation buffer in each study.
  • mice were randomized into groups of 10 animals each, 7 days post MC-38-5AG cell implantation according to tumor volume (TV), with mean right and left TV of 26 mm3 and 24 mm 3 , respectively.
  • mice received intratumoral injections of vehicle or mJP- OV-2.
  • mice received intraperitoneal (IP) injections of anti-PD-1 antibody RMP1 14 according to the study design.
  • IP intraperitoneal
  • Table 15 A summary of the study design described above is provided in Table 15.
  • EXAMPLE 2 PHASE 1 STUDY OF INTRATUMORAL ADMINISTRATION OF JP-OV-2, AN ONCOLYTIC VIRUS, AS MONOTHERAPY AND IN COMBINATION FOR ADVANCED SOLID TUMORS
  • This example describes a Phase 1 study of intratumoral administration of JP-OV-2, an oncolytic virus, as monotherapy and in combination with a PD-1 antibody, cetrelimab, for advanced solid tumors.
  • JP-OV-2 is a next-generation recombinant HSV Type-l-based oncolytic virus (OV) that has been modified to 1) reduce innate and adaptive antiviral host responses that shut down viral replication to allow greater lysis of target tumor cells and 2) to enhance all stages of the adaptive immune response to the cancer cells through expression of multiple synergistic immune stimulatory payloads.
  • OV HSV Type-l-based oncolytic virus
  • Cetrelimab is a fully human IgG4K monoclonal antibody that binds to PD-1. Cetrelimab comprises a heavy chain and a light chain, wherein the heavy chain of the cetrelimab comprises a heavy chain variable region (VH) comprising a CDR-H1, CDR-H2, and CDR-H3 of a VH comprising the amino acid sequence set forth in SEQ ID NO:400, and the light chain of the cetrelimab comprises a light chain variable region (VL) comprising a CDR-L1, CDR-L2, and CDR-L3 of a VL comprising the amino acid sequence set forth in SEQ ID NO:404.
  • VH heavy chain variable region
  • CDR-H2 CDR-H2
  • CDR-H3 of a VH comprising the amino acid sequence set forth in SEQ ID NO:400
  • VL light chain variable region
  • Cetrelimab comprises a VH comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 401, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 402, a CDR- H3 comprising the amino acid sequence set forth in SEQ ID NO: 403; and/or a VL comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 405, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 406, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 407.
  • the first expansion cohort will consist of participants with relapsed (Cohort A) and treatment-naive (Cohort B) metastatic NSCLC.
  • JP-OV-2 is given via intratumoral injection Q2W.
  • Dose Escalation of JP-OV-2 begins at a starting dose of 10 6 PFU/mL (number of viral plaque-forming units per mL of solution) with the number of mLs administered dependent on the size of the tumor.
  • 1 or more lesions may be injected, and the maximum volume administered across all injected lesions is 10 mL.
  • the first dose of JP-OV-2 is administered to each participant as a monotherapy and then in combination with cetrelimab beginning with the second dose of JP-OV-2.
  • the first participant treated at a dose level is evaluated for at least 5 days prior to treating subsequent participants.
  • Cetrelimab is administered at a dose of 480 mg IV once every 4 weeks (Q4W). Once a dose level of JP-OV-2 is determined by the study evaluation team (SET) to be safe as a monotherapy, additional participants may be enrolled to the next higher dose level of JP-OV-2 monotherapy as guided by the BOIN design. Combination of JP-OV-2 at the higher dose level with cetrelimab will not begin until the SET confirms the safety of the JP-OV-2 and cetrelimab combination at the previous dose level during a 14-day combination DLT observation period.
  • a dosing table (Table 16) is provided below.
  • Core needle biopsies may be obtained from on-treatment tumor samples during Part 1 to assess changes in the tumor microenvironment. At least 1 injected and at least 1 uninjected lesion prior to the fourth dose of JP-OV-2 will be obtained. The injected lesion should be the same as the one biopsied at baseline.
  • DLT dose limiting toxicity
  • additional dose levels may be enrolled per SET decision, to evaluate alternative JP-OV-2 dosing frequencies or ratios of tumor volume to injected JP-OV-2 volume.
  • JP-OV-2 dosing regimens dose intensification
  • treatment will be initiated and continued with JP-OV-2 in combination with cetrelimab so that participants will receive combination treatment starting with the first dose.
  • JP-OV-2 dose intensification
  • up to 8 treatments with JP-OV-2 may be given. Additional treatments, up to 8 more doses, may be given with sponsor approval on a case-by-case basis and upon satisfying defined criteria.
  • the first alternative (intensified) dosing regimen (Table 17) will evaluate JP-OV-2 administration on Days 1 and 8 of each 28-day cycle of cetrelimab.
  • a second intensified regimen may be evaluated (both doses within first 7 days of each 28-day cycle of cetrelimab) based upon safety, and PK data, to optimize intratumoral payload effect (Table 18).
  • the SET may decide to evaluate higher ratios of JP-OV-2 per tumor volume only after the first 3 dose levels have been evaluated and the SET have agreed that it is safe to do so.
  • Higher ratios of injected JP-OV-2 to tumor volume will be achieved by limiting the largest injected lesion diameter to 4 cm (vs >5 cm per Table 20) to receive the full 10 mL target JP-OV-2 injection.
  • the higher injected JP-OV-2 volumes per lesion size are provided in Table 19. These injection volumes may be evaluated on Q2W schedule or alternative schedules, provided the schedule has been used with the dose volumes defined in Table 20.
  • the volume listed in the table should be the full amount prescribed for lesions in each size category, “up to” denotes the possibility that the full amount may not be feasible to administer to the lesion. This may occur, for example, if the lesion has decreased in size since the last disease assessment or if the pressure required to administer the study treatment becomes prohibitive in the opinion of the treating physician.
  • “Large or spherical superficial lesions may be injected using the visceral injection volumes.
  • Part 2 of the study will further evaluate the safety and tolerability of JP-OV-2 (used at the doses and regimens identified in Part 1) in combination with a PD-1 antibody and will also assess the preliminary efficacy of the treatment regimen.
  • Cohorts A and B will evaluate treatment with JP-OV-2 in combination with cetrelimab at the dose identified in Part 1 in participants with metastatic NSCLC who have progressed after treatment with anti-PD-1 and chemotherapy.
  • treatment is initiated and continued with combination of JP-OV-2 in combination with cetrelimab so that participants will receive combination treatment starting with the first dose.
  • Cohort B will be initiated after review of safety data from the first 3 participants in Cohort A treated with the combination treatment for at least 14 days.
  • Cohort A of Part 2 will consist of participants with advanced metastatic NSCLC who have progressed after treatment with anti-PD-1 and chemotherapy and have a PD-L1 ⁇ 1% tumor proportion score.
  • Cohort B of Part 2 will consist of participants with advanced metastatic NSCLC who have never been treated for their metastatic disease and a PD-L1>50% tumor proportion score. Up to 30 participants may be treated in each Cohort A and B.
  • JP-OV-2 is administered Q2W via intratumoral injection up to a maximum volume of 10 mL divided within the selected lesions.
  • the SET may decide to investigate additional dosing regimens as described previously. Although doses may be skipped based on clinical feasibility and medical condition of the participant, every effort should be made to ensure that the participants receive all scheduled doses prior to the first disease assessment to maximize probability of clinical benefit. Injections may be performed by 3 approaches depending on locations of lesions and part of study enrollment. Different approaches may be utilized at the same or different visits depending on the location of all injectable lesions and clinical feasibility.
  • JP-OV-2 can be administered via a superficial approach with injection under direct visualization/palpation into a cutaneous, subcutaneous, or nodal lesion (ultrasound guidance optional to facilitate/monitor but not required);
  • JP-OV-2 can be administered via a percutaneous approach for lesions accessible via imaging guidance (such as transthoracically accessible lung lesions; transabdominally accessible liver lesions, subcutaneous soft tissue lesions etc.); and,
  • JP-OV-2 can be administered via an endobronchial approach for lung lesions and lymph nodes accessible via a bronchoscope.
  • cetrelimab may be administered on the next day.
  • Treatment may be continued for up to a maximum of 8 doses of JP-OV-2 and 2 years with cetrelimab unless discontinuation criteria are reached earlier (confirmed radiographic disease progression, unequivocal clinical disease progression, unacceptable toxicity, withdrawal of consent, investigator decision, the participant becomes pregnant, or the participant is lost to follow- up). Treatment may be continued after confirmed radiographic disease progression if deemed by the investigator to be in the best interest of the participant and after obtaining approval from the physicians involved in participant’s treatment and from the sponsor medical monitor. Continuation or reinitiation of JP-OV-2 up to an additional 8 doses may be permitted during cetrelimab treatment after consultation with the sponsor medical monitor.
  • Injection volume for each lesion is based on the longest diameter of the lesion (including lymph nodes) per the most recent disease evaluation radiographic scan prior to scheduled treatment and following the doses provided in Table 17.
  • the maximum injection volume per administration/treatment cycle is 10 mL.
  • This volume may be divided into aliquots and injected into multiple tumor lesions via the appropriate injection route (superficial, percutaneous, or endobronchial).
  • injection route superior, percutaneous, or endobronchial
  • As many lesions as possible should be injected keeping the total injected volume as close to 10 mL as feasible.
  • For injections into visceral lesions no more than 3 lesions within a single solid organ should be injected at a single administration visit unless discussed with the sponsor.
  • CT or CBCT intraprocedural imaging
  • Participants may receive injections in more than one organ/site of disease and the intratumoral injection procedure is determined by the lesion type and location. At each treatment visit, one or more lesions may be injected, and the maximum total JP-OV-2 dose volume injected across all lesions is 10 mL, with injection volume per lesion guided by Table 17. For injections in the lung, only lesions in one hemithorax (one lung) can be injected per administration.
  • the number of lesions and organ sites to be injected is determined based on the number and size of accessible lesions to achieve a total dose volume of 10 mL injected in all lesions at each JP-OV-2 treatment visit.
  • At least 1 lesion selected as an uninjectable target lesion for RECIST v1.1 assessment should not be injected, unless all other lesions become uninjectable. In this case, sponsor must be consulted prior to injecting the remaining uninjected target lesion.
  • each lesion be injected with the maximum amount recommended per Table 17 before moving to the next lesion, subject to tumor specific limitations. Clusters of lesions may be considered as a single lesion for injection.
  • Lesions for initial injections should be selected as to increase the feasibility for biweekly injection, at least through the first disease assessment, to maximize possibility of achieving clinical benefit. For the first administration visit, it is recommended to inject the largest injectable lesion that is clinically appropriate. For Part 2, the largest injectable lung lesion should be prioritized for injection via endobronchial or transthoracic approach if feasible. Primary lung lesion and affected measurable lymph nodes should be injected next. Inject any remaining injectable lesions based on the lesion size until the maximum volume of 10 mL has been injected or no injectable lesions remain.
  • a superficial lesion is a cutaneous, subcutaneous, or nodal lesion that can be accessed in the clinic under direct visualization/palpation (ultrasound guidance optional to facilitate/monitor but not required).
  • Visceral lesions or deep nodal lesions require imaging guidance (ultrasound, CBCT, or CT) for injection. Visceral lesions should be safe to inject in the opinion of the investigator (eg, avoiding lesions with major airway or blood vessel invasion). Lesions in locations with a history of poor wound healing should be avoided. Lesions in previously irradiated fields or other risk factors for poor wound healing (eg, poor vascularization) should be discussed with sponsor’s medical monitor prior to administration. For injections into visceral lesions, no more than 3 lesions within a single solid organ should be injected at a single administration visit unless discussed with the sponsor.
  • injections into the lung may involve endobronchial injection via a bronchoscope or percutaneous image-guided injections for lesions that are not accessible by a bronchoscope, based on the clinical decision of the performing physician.
  • Each potential participant must satisfy all of the following criteria to be enrolled in the study: [0334] 1. Be >18 years of age (or the legal age of majority in the jurisdiction in which the study is taking place, whichever is greater) at the time of informed consent.
  • Part 1 Individuals with a diagnosis of advanced or metastatic solid tumor (except tumors of the CNS), who have previously received available standard therapy and progressed, or cannot tolerate standard therapy, or for whom there is no standard of care per regional guidelines. Individuals who have previously refused standard therapy in consultation with their health care professional may participate as long as documentation of the medical endorsement for their decision is provided.
  • injectable tumor >1 cm in longest diameter (or shortest diameter for lymph nodes) or injectable tumors that in aggregate are >1 cm in the longest diameter, which is/are also measurable by RECIST v1.1.
  • injectable lesion considerations eg, injectable lesion must not be invading or in close proximity to major or large blood vessels or invading major airways. Eligibility for intratumoral treatment must be reviewed with all physicians to be involved in the participant’s treatment.
  • Part 2 Have at least 1 measurable lesion that will not be injected during the study unless approved by the Sponsor.
  • Participants with known hepatic metastases are eligible if they have: AST ⁇ 5 x ULN, ALT ⁇ 5 x ULN, and Total bilirubin ⁇ 3 x ULN (isolated total bilirubin ⁇ 3 x ULN with conjugated [direct] bilirubin ⁇ 1.5 x ULN is allowed for those participants with known congenital nonhemolytic hyperbilirubinemias).
  • Eligible participants include: (a) female participants not of childbearing potential.
  • a participant of childbearing potential must have a negative highly sensitive serum (eg, ⁇ -hCG) pregnancy test at screening and within 72 hours of the first dose of study treatment and must agree to further serum or urine pregnancy tests during the study.
  • ⁇ -hCG highly sensitive serum
  • a participant using oral contraceptives must use an additional barrier contraceptive method.
  • a participant must agree not to donate gametes (ie, eggs or sperm) or freeze for future use for the purposes of assisted reproduction during the study and for a period of 5 months after receiving the last dose of study treatment. Participants should consider preservation of gametes prior to study treatment as anticancer treatments may impair fertility.
  • a participant must wear a condom when engaging in any activity that allows for passage of ejaculate to another person during the study and for 5 months after receiving the last dose of study treatment.
  • a participant who is vasectomized must still use a condom (with or without spermicide).
  • TSH thyroid stimulating hormone
  • T3 total or free
  • T4 free thyroxine
  • Active autoimmune disease that requires systemic immunosuppressive medications (eg, chronic corticosteroid, methotrexate, or tacrolimus) within the 12 months prior to signing consent.
  • systemic immunosuppressive medications eg, chronic corticosteroid, methotrexate, or tacrolimus
  • Venous thromboembolic events eg, pulmonary embolism
  • uncomplicated (Grade ⁇ 2) deep vein thrombosis is not considered exclusionary.
  • cardiovascular disease including any of the following within 6 months prior to signature of informed consent: Myocardial infarction, severe or unstable angina, or coronary artery bypass surgery, clinically significant arrhythmias (eg, ventricular arrhythmias or atrial fibrillation with uncontrolled heart rate), congestive heart failure (NYHA class III/V), cerebrovascular accident, transient ischemic attack, or other arterial thromboembolic event, or Myocarditis.
  • arrhythmias eg, ventricular arrhythmias or atrial fibrillation with uncontrolled heart rate
  • congestive heart failure NYHA class III/V
  • cerebrovascular accident e.g, transient ischemic attack, or other arterial thromboembolic event, or Myocarditis.
  • Participants with recent or planned surgical procedures utilizing only local anesthesia may participate.
  • Part 1 and Part 2 Cohort A Prior treatment with an HSV-based oncolytic virus for the treatment of metastatic disease.
  • Part 2 Cohort B Any prior treatment for metastatic disease with the exception of a single dose of anti-PD(L)-1 agent administered prior to the first dose of study treatment (see Inclusion Criterion 2). Chemotherapy for non-metastatic NSCLC is allowed as long as metastatic disease did not develop within one year following this treatment. Radiation therapy or surgery for non- metastatic disease are allowed as long as the windows specified in other exclusion criteria are respected.
  • Part 1 and Part 2 Cohort A Prior anti-PD(L)-l or anti-CTLA-4 therapy within 4 weeks or other anti cancer therapy within 14 days before the first dose of study treatment.
  • the primary objective of Part 1 is to determine a safe, tolerable, and feasible dose of intratumorally delivered JP-OV-2 and the recommended dose(s) and regimen(s) in combination with cetrelimab in participants with advanced solid tumors.
  • the incidence and severity of AEs, including DLTs, are used to determine the dose(s) and regimen(s) of JP-OV-2 as a monotherapy, and in combination with cetrelimab, to evaluate in Part 2.
  • the primary objective of Part 2 (dose expansion) is to further characterize the safety and tolerability of JP-OV-2 at the selected dose(s) in combination with a PD-1 antibody in select tumor populations.
  • the first 2 cohorts in Part 2 will evaluate the safety of JP-OV-2 at the selected dose(s) in combination with cetrelimab in participants with previously treated metastatic NSCLC with no PD-L1 expression (PD-L1 ⁇ 1%) at the time of original diagnosis (Cohort A), and in treatment-naive participants with metastatic NSCLC with high PD-L1 expression (PD-L1 ⁇ 50%, Cohort B).
  • Secondary objectives in both parts of the study include determination of the biodistribution of JP-OV-2, viral shedding, immunogenicity, cetrelimab PK, and preliminary clinical activity of JP-OV-2 in combination with cetrelimab.
  • Dose Escalation (Part 1): Recommended dose(s) of JP-OV-2 alone and in combination with cetrelimab may be identified such that the isotonic estimate of the DLT rate for monotherapy and the combination is less than or equal to the target rate of 33%.
  • Part 1 Number of Participants with Dose-Limiting Toxicity (DLT) [ Time Frame: Up to 5 years ].
  • the DLTs are specific adverse events and are defined as any of the following: non- hematological toxicity and hematologic toxicity.
  • AEs Number of Participants with Adverse Events (AEs) by Seventy [ Time Frame: From first dose up to 100 days after last dose of study treatment (up to 5 years) ].
  • An adverse event is any untoward medical occurrence in a clinical study participant administered a pharmaceutical (investigational or non-investigational) product. An adverse event does not necessarily have a causal relationship with the treatment. Severity will be graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 5.0. Seventy scale ranges from Grade 1: mild, Grade 2: moderate, Grade 3: severe. Grade 4: life-threatening, and Grade 5: death related to adverse event.
  • OR Percentage of Participants With Objective Response
  • OR is defined as the percentage of participants who have best response of Complete Response (CR) or Partial Response (PR) according to response evaluation criteria in solid tumors (RECIST) v1.1.
  • DC Percentage of Participants With Disease Control
  • Parts 1 and 2 Duration of Response (DOR) [ Time Frame: Up to 5 years ]. DOR will be calculated among responders from the date of initial documentation of a response to the date of first documented evidence of relapse according to RECIST v1.1, or death due to any cause, whichever occurs first.
  • PFS Progression Free Survival
  • OS Overall Sunrival
  • Parts 1 and 2 Number of JP-OV-2 Genome Copies per Milliliter [ Time Frame: Up to 5 years ]. Viral genome copies of JP-OV-2 collected from samples (that is, blood, urine, oral mucosa, injection sites, and dressings) will be determined by quantitative polymerase chain reaction (qPCR) assays.
  • qPCR quantitative polymerase chain reaction
  • Parts 1 and 2 Payload Concentrations of JP-OV-2 [ Time Frame: Up to 2 years ]. Blood samples will be collected to characterize JP-OV-2 payload concentrations in blood and tumor and will be analyzed using immunoassay.
  • Parts 1 and 2 Number of Participants with JP-OV-2 Antibodies [ Time Frame: Up to 2 years ]. Antibodies against JP-OV-2 encoded payloads and against herpes simplex virus type-1 (HSV-1) will be analyzed.
  • HSV-1 herpes simplex virus type-1
  • Baseline disease burden is assessed using CT scans of the chest, abdomen, and pelvis, plus other areas of known disease involvement as appropriate, with IV contrast. Participants who are intolerant of IV contrast agents may have CT scans performed with oral contrast and the reason for not using IV contrast are documented in source documents. Subsequent efficacy evaluations during the study will include radiographic imaging of all disease sites documented at baseline.
  • Magnetic resonance imaging may be used to evaluate sites of disease that cannot be adequately imaged using CT scan.
  • an MRI it must be the imaging technique used to assess disease at baseline and at all subsequent response evaluations.
  • MRI assessments do not replace the required chest, abdomen, and pelvic CT scans, unless CT scan is contraindicated. Brain MRI is required only if clinically indicated. CT scan of the head can be used if MRI is contraindicated.
  • Serum samples are collected to assess the pharmacokinetics and ADA of cetrelimab in this participant population.
  • Immunogenicity assessments are conducted to determine the presence of antibodies against viral antigens expressed by JP-OV-2, the 4 encoded payloads, and cetrelimab. Other analyses may be performed to further characterize the immunogenicity of JP-OV-2, its payloads, and cetrelimab. Samples collected for immunogenicity analyses may additionally be used to evaluate safety or efficacy aspects that address concerns arising during or after the study period. Genetic analyses will not be performed on these serum samples.
  • ORR is tabulated with its 95% exact confidence interval. In addition, the number and percentage of participants in each response category are tabulated. For the calculation of ORR, the treated participants who are not evaluable for response are listed as such and considered as non-responders. Both the overall ORR as well as the separate ORRs of injected and uninjected lesions are calculated. [0407] For DOR, PFS, and OS the Kaplan-Meier method is used for descriptive summaries (eg, median and Kaplan-Meier curve).
  • JP-OV-2 The safety of JP-OV-2 is assessed by physical examinations, ECOG performance status, vital signs, clinical safety laboratory tests, viral shedding swabs, pregnancy testing, and monitoring for AEs, including DLTs.
  • the dose escalation study evaluated the treatment of three escalating dose levels of JP- OV-2 administered intratumorally as monotherapy for the initial dose, followed by administration in combination with systemic anti-PD1 therapy (cetrelimab) for subsequent doses. This study involved patients with advanced solid tumors who had experienced relapse after all approved therapies.
  • the swim lane plot (FIG. 4) illustrates the duration of treatment (represented by the length of the horizontal bars) and the best clinical response (shown as white circles) for individual patients treated at each dose level.
  • the assessment of response was conducted according to oncology standards using RECIST v1.1, which defines a response as a reduction in total measurable tumor burden by at least 30%.
  • Clinical benefit was defined as stable disease according to RECIST for a minimum of 12 weeks.
  • the repeated intratumoral doses of JP-OV-2, administered every two weeks, were well tolerated.
  • Other tumor types showing benefit included colon cancer (Patient 100004 - 12 weeks), thymic cancer (Patient 100008, 28 weeks), pancreatic cancer (Patient 100009, 12 weeks), and anal cancer (Patient 100022, 16 weeks).
  • FIG. 5A presents a waterfall plot that shows the post-treatment change in total measurable tumor burden per standard oncology guidelines (RECIST v1.1), with vertical bars representing individual patients.
  • This figure demonstrates a dose-dependent effect of JP-OV-2 on tumor growth control, with effects increasing with ascending doses from left to right; the 10° dose is shown in the first third, the 10'' dose in the second third, and the 10 8 dose in the final third of FIG. 5 A.
  • the 10° dose is shown in the first third
  • the 10'' dose in the second third the 10 8 dose in the final third of FIG. 5 A.
  • 10 8 dose-dependent effect of JP-OV-2 on tumor growth control, with effects increasing with ascending doses from left to right
  • the 10° dose is shown in the first third
  • the 10'' dose in the second third the 10 8 dose in the final third of FIG. 5 A.
  • At the top dose level of 10 8 two patients with advanced melanoma achieved a ⁇ 30% reduction in tumor burden, qualifying as a
  • FIG. 6A sho ws the quantity of each of the payload proteins detected in these tumor tissues. Pay load proteins were identified in all six tested tissue samples, with all four payloads detectable in four out of six samples and three payloads detected in the remaining two samples. Importantly, the relative levels of payload proteins detected were similar to those observed in preclinical mouse models (FIG. 6B). A direct correlation was found between the amount of payload protein and serum levels of interferon-y (Fig. 3C), indicating immune activation resulting from the effects of the immune payloads.
  • FIG. 7 provides data for a responding patient at the top dose level (patient 100027).
  • patient 100027 This patient had metastatic melanoma that was unresponsive to three prior immunotherapy treatments, including anti-PD1 and anti-CTLA-4 therapies. While melanoma typically responds to immunotherapy, patients with refractory disease typically have poor outcomes, with no effective therapies available, and palliative treatments usually lead to disease progression within 2-3 months. Such tumors are considered immunologically “cold” and are not expected to respond to available immunotherapeutic options. Following treatment with JP-OV-2, this patient exhibited a response in both injected and uninjected lesions and has maintained this response for over four months as of the latest data review.
  • the figure includes CT scan images of a representative injected and uninjected lesion (FIG.
  • FIG. 8 illustrates serum levels of interferon-y at various time points during treatment for patients treated at each JP-OV-2 dose level. Dose-proportional increases in interferon-y levels were observed across the three evaluated doses of JP-OV-2, indicating a dose-dependent effect of JP- OV-2 and its encoded payloads on immune activation in cancer patients. Notably, HSV seronegative patients exhibited higher levels after the first dose, which decreased by the second dose to levels comparable to seropositive patients, correlating with seroconversion.
  • T 6 examine the hypothesis that the injection of JP-OV-2 induces both direct and indirect effects mediated by the encoded immune payloads, we evaluated the immune microenvironment in paired tumor tissue samples collected from a lesion before treatment and another sample from the same lesion after three injections of JP-OV-2 (FIG. 9). These samples were obtained from a patient diagnosed with mesothelioma, a disease known for its resistance to immunotherapy and classified as having "cold" immune biology. Consistent with this characterization, the patient previously underwent treatment with anti-PD1 and anti-CTLA-4 therapies but experienced limited benefits before enrolling in this study. The patient was treated with JP-OV-2 at the second dose level, with sequential injections at two different sites when the first site showed response. The patient demonstrated a reduction in total tumor burden, achieving sustained disease control for seven months while receiving a total of twelve injections of JP-OV-2 at two- week intervals, without any skipped doses.
  • JP-OV-2-encoded immune payloads are expressed at the injection sites, enhancing the immune anti-tumor response not only at the injection site but also at uninjected sites when combined with the anti-PD1 agent cetrelimab, resulting in clinical responses in both injected and uninjected tumors.

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Abstract

Provided herein are methods for treating cancer in an individual comprising administering an oncolytic virus in combination with a PD-1 antibody. The provided methods trigger an abscopal response in distant tumors, facilitating the systemic activation of the immune system to identify and target untreated tumor sites. By harnessing the dual mechanisms of oncolytic viruses and immune checkpoint inhibition, the methods provided herein enhance overall treatment efficacy, offering a novel approach to the treatment of various types of malignancies.

Description

ONCOLYTIC VIRUSES EXPRESSING IMMUNOMODULATORS AND USE FOR TREATING ADVANCED SOLID TUMOR
TECHNICAL FIELD
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of United States Provisional Application Serial Numbers 63/562,793 and 63/562,795, both filed on March 8, 2024. The entire contents of these applications are incorporated herein by reference in their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on February 28, 2025, is named JBI6890WOPCTl_SL.xml and is 235,867 bytes in size.
TECHNICAL FIELD
[0003] The present disclosure relates to methods of treating cancer comprising oncolytic viruses derived from herpes simplex virus 1 (HSV-1), administered in combination with a PD-1 antibody.
BACKGROUND
[0004] Oncolytic virus therapy is a form of immunotherapy that exploits the cytotoxic and/or vector ability of viruses to selectively target and destroy tumor cells. Oncolytic virus therapy can also work to stimulate immune responses against a target tumor or tumors distal to a target tumor. Safety issues limited the use of live, infectious viruses in cancer patients, but the development of robust genetic engineering has allowed the field to mature by the development of improved viruses (Kelly and Russell, Mol. Ther. 2007 Apr; 15(4): 651-9). Oncolytic viruses may be engineered to have enhanced selectively for tumor cells (for example, by enhanced cytotoxicity in cancer cells and/or reduced cytotoxicity in normal cells) and to express therapeutic payloads, such as immunostimulatory proteins.
[0005] Herpes simplex viruses (HSV) are candidates for additional oncolytic virus development. HSV has a broad host cell range in humans, a short replication cycle, a large genome which is amenable to multiple payload genes, and effective antiviral options to control infection (Sanchala et al., Front. Pharmacol. 2017;8:270). Next generation oncolytic viruses, including those derived from HSV, that exhibit increased safety and increased efficacy are therefore needed in the art. Additionally, whether oncolytic viruses could provide efficacious cancer therapy in combination with other immunotherapy based approaches is poorly understood. Provided herein are methods and compositions that address such and other needs.
[0006] All references cited herein, including patent applications, patent publications, and scientific literature, are herein incorporated by reference in their entirety, as if each individual reference were specifically and individually indicated to be incorporated by reference
SUMMARY
[0007] Provided herein are methods of treating cancer in an individual comprising administering an oncolytic virus in combination with a PD-1 antibody to the individual. In some embodiments, the oncolytic viruses provided herein trigger an abscopal response to a distant tumor and/or cause an immunological memory of a tumor.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] The present application can be understood by reference to the following description taken in conjunction with the accompanying figures.
[0009] FIGS.1A-1B are diagrams of the genome structure and transgene cassettes of the mouse surrogate virus, mJP-OV-2 and the human virus JP-OV-2. As shown in FIG. 1A, the mouse surrogate virus mJP-OV-2 was engineered to express the anti-CTLA-4 antagonist (mαCTLA-4), CD40 agonist (mCD40ag), and IL- 12 (mscIL-12) payloads from the US 10- 12 locus, and hFLT3L and UL49.5 from the γ34.5 locus. The virus expresses codon-optimized US11 (hCoUS11) using the US 12 immediate early promoter and endogenous US 11 using late US 11 promoter. As shown in FIG. IB, the human virus JP-OV-2 was engineered to express the anti-CTLA-4 antagonist (hαCTLA-4), CD40 agonist (hCD40ag), and IL- 12 (hscIL-12) payloads from the US 10- 12 locus, and hFLT3L and UL49.5 from the γ34.5 locus. The virus expresses codon-optimized US 11 (hCoUS 11) using the US 12 immediate early promoter and endogenous US 11 using late US 11 promoter. “S” shown in the hexagon represents a stop codon between hCoUS 11 and US 12, which inhibits US12 expression. US12 in grey indicates that the gene does not express. IRL, internal repeat long; IRS, internal repeat short; TRL, terminal repeat long; TRS, terminal repeat short; UL, unique long; US, unique short.
[0010] FIGS. 2A-2C show the effect of treatment with an oncolytic HSV-1 virus in combination with a PD-1 inhibitor in a mouse tumor model system. FIG. 2A shows a schematic for the experimental design. On Day 0, MC-38-5 AG tumor cells were implanted bilaterally on each flank of mice (n=10/group). Mice were treated intratumoral on Days 8, 11, and 14 with vehicle or mJP- OV-2 (arrows). Some groups were treated intraperitoneal (IP) on Days 8, 12, 15, and 19 with anti- PD-1 RMP1 14. FIG. 2B shows the tumor volume for the treated and untreated tumors and graphed as the mean ± SEM. Tumor growth of the treatment groups was compared statistically with growth of the vehicle control group over time to Day 29. FIG. 2C shows survival of the treatment groups compared with that of the vehicle control group for oncolytic HSV-1 virus treatment in combination with an anti-PD-1 antibody. * p<0.05, ** p<0.01, *** p<0.001. IT, intratumoral; SEM, standard error of the mean.
[0011] FIGS. 3A-3C illustrate the design of a Phase 1 study. FIG. 3 A shows a schematic overview of the study including the dose escalation analysis in Part 1 (right) and the dose expansion analysis in part 2 (left). a Depending on combination partner and safety profile of JP-OV-2, subsequent combinations may use the identified monotherapy dose of JP-OV-2 and established doses of cetrelimab without pursuing dose escalation. FIG. 3B shows the administration schedule for Part 1. FIG. 3C shows the administration schedule for Part 2. a Refer to Example 2 for full treatment duration description. b Additional treatments, up to 8 more doses, may be given with sponsor approval on a case by case basis. c Doses may be skipped based on clinical feasibility and medical condition of the patient.
[0012] FIG. 4 presents a swim lane plot that illustrates the duration of treatment, indicated by the length of the horizontal bars, alongside the best clinical response for individual patients at each dose level, represented by white circles. The response is evaluated according to oncology standards, specifically using RECIST v1.1 criteria, which classifies a response as a reduction in total measurable tumor burden of at least 30%. Additionally, clinical benefit is defined as the presence of stable disease according to RECIST for a minimum duration of 12 weeks. The bars without any white circles have not had a disease evaluation.
[0013] FIG. 5 A presents a waterfall plot that displays the post- treatment change in total measurable tumor burden according to standard oncology guidelines (RECIST v1.1). The vertical bars represent individual patients and illustrate a dose-dependent effect of JNJ916 on tumor growth control, with effects increasing from left to right across the plot. The first third of FIG. 5 A depicts the 106 dose, the second third shows the 107 dose, and the final third represents the 108 dose.
[0014] FIG. 5B highlights tumor shrinkage in uninjected lesions (represented by light bars).
[0015] FIG. 6A illustrates the quantities of each payload protein detected in tumor tissues collected from patient samples, as assessed using Meso Scale Discovery (MSD) assay.
[0016] FIG. 6B illustrates the clinical payload expressions in preclinical mouse models.
[0017] FIG. 6C shows the correlation between the tumoral payload levels and interferon-y.
[0018] FIGs. 7A, 7B, and 7C display the data for a responding patient at the highest dose level. The drawings presented include CT scans of a representative injected lesion and an uninjected lesion (FIG. 7A), quantification of serum levels of JP-OV-2-encoded immune payloads (FIG. 7B), and the corresponding effects on immune activation, illustrated by serum interferon-γ levels (FIG. 7C).
[0019] FIG. 8. displays serum levels of interferon-y at various time points during treatment for patients treated at each JP-OV-2 dose level.
[0020] FIG. 9 depicts the immune microenvironment in paired tumor tissue samples collected from the same lesion. One sample was obtained before the injection of JP-OV-2, and the other was collected after the administration of three JP-OV-2 injections.
DETAILED DESCRIPTION
[0021] The development of cancer immunotherapies that harness the immune system to restore anti-tumor immunity offers to significantly improve the way in which clinicians treat cancer. However cancers often develop immunosuppressive microenvironments, significantly limiting the efficacy of current cancer immunotherapies.
[0022] The present disclosure provides methods of treating solid tumors in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus and a PD-1 antibody to the individual. The oncolytic virus disclosed herein is armed with four immunomodulatory pay loads: human fms like tyrosine kinase 3 ligand (hFLT3L), human cluster of differentiation (hCD)40 agonist (ag), antibody to human cytotoxic T lymphocyte-associated protein 4 (αCTLA4), and human single-chain (hsc) interleukin (IL) 12. These immunomodulatory payloads, when expressed in tumor cells, significantly alter the tumor microenvironment to be more favorable for immune cell function. Delivery of a PD-1 antibody further stimulates the immune response. Prior to the present disclosure, few therapies successfully utilized oncolytic viruses to modulate the tumor microenvironment.
[0023] As demonstrated herein, treatment with oncolytic herpes simplex type 1 (HSV-1) virus and a PD-1 antibody causes direct and abscopal tumor growth inhibition that results in prolonged survival. Such treatment also produces durable antitumor responses, demonstrating the development of a proficient and sustained antitumor response.
[0024] The following description sets forth exemplary methods, parameters and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.
I. Definitions
[0025] In order that the present disclosure can be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.
[0026] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub- combination. All combinations of the embodiments pertaining to particular method steps, reagents, or conditions are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed.
[0027] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
[0028] Reference to “about” a value or parameter herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) aspects that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.
[0029] The term "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
[0030] It is understood that aspects and embodiments of the invention described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments.
[0031] As used herein, the terms “including,” “containing,” and “comprising” are used in their open, non-limiting sense.
[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei- Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.
[0033] The term “antibody” herein is used in the broadest sense and encompasses various antibody structures (immunoglobulin molecules, fragments of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen under typical physiological conditions), including but not limited to monoclonal antibodies, 4-chain antibodies (such as IgG antibodies), heavy chain antibodies, and antibody fragments thereof so long as they exhibit the desired antigen-binding activity. The term “4-chain antibody” is used herein to refer to an antibody or antigen-binding fragment having two heavy chains and two light chains. The term “heavy chain antibody,” also known as “heavy chain-only antibody” or “HCAb” refers to a functional antibody, which comprises two heavy chains, but lacks two light chains usually found in 4-chain antibodies. Camelid animals (such as camels, llamas, or alpacas) are known to produce HCAbs.
[0034] An "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities. An isolated antibody that binds specifically to an antigen can, however, have cross-reactivity to other antigens, such as homologous antigens from other species. Moreover, an isolated antibody can be substantially free of other cellular material and/or chemicals.
[0035] “Antibody fragments” comprise a portion of an antibody, preferably the antigen binding or variable region of the antibody. Examples of antibody fragments include VHHs, single-domain antibodies, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see US Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. The term “constant domain” refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant domain contains the CH1, CH2 and CH3 domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain.
[0036] As used herein, the terms “binding”, "binds" or "specifically binds" in the context of the binding of an antibody to a pre-determined antigen typically is a binding with an affinity corresponding to a KD of about 106 M or less, e.g. 107 M or less, such as about 108 M or less, such as about 109 M or less, about 1010 M or less, or about 1011 M or even less when determined by for instance BioLayer Interferometry (BLI) technology in a Octet HTX instrument using the antibody as the ligand and the antigen as the analyte, and wherein the antibody binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100- fold lower, for instance at least 1,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than its KD of binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen. The amount with which the KD of binding is lower is dependent on the KD of the antibody, so that when the KD of the antibody is very low, then the amount with which the KD of binding to the antigen is lower than the KD of binding to a non-specific antigen may be at least 10,000-fold (that is, the antibody is highly specific).
[0037] The term "KD" (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Affinity, as used herein, and KD are inversely related, that is that higher affinity is intended to refer to lower KD, and lower affinity is intended to refer to higher KD. [0038] A “CDR” refers to one of three hypervariable regions (H1, H2, or H3) within the non- framework region of the immunoglobulin (Ig or antibody) VH P-sheet framework, or one of three hypervariable regions (L1, L2, or L3) within the non-framework region of the antibody VL β-sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined by, for example, Kabat as the regions of most hypervariability within the antibody variable (V) domains. Kabat et al., J. Biol. Chem. 1977, 252, 6609-6616; Kabat, Adv. Protein Chem. 1978, 32, 1-75. CDR region sequences also have been defined structurally by Chothia as those residues that are not part of the conserved P-sheet framework, and thus are able to adapt different conformations. Chothia and Lesk, J. Mol. Biol. 1987, 196, 901-917. Both terminologies are well recognized in the art. CDR region sequences have also been defined by AbM, Contact and IMGT. The positions of CDRs within a canonical antibody variable region have been determined by comparison of numerous structures. Al-Lazikani et al., J. Mol. Biol. 1997, 273, 927-948; Morea et al., Methods. 2000, 20, 267-279. Because the number of residues within a hypervariable region varies in different antibodies, additional residues relative to the canonical positions are conventionally numbered with a, b, c and so forth next to the residue number in the canonical variable region numbering scheme. Al-Lazikani et al., supra (1997). Such nomenclature is similarly well known to those skilled in the art.
[0039] The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is often defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxy 1-terminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.
[0040] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
[0041] The term “hypervariable region” or “HVR” as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence (“complementarity determining regions” or “CDRs”) and/or form structurally defined loops (“hypervariable loops”) and/or contain the antigen-contacting residues (“antigen contacts”). Generally, 4-chain antibodies and antigen- binding antibody fragments thereof comprise six HVRs: three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). Generally, heavy-chain antibodies comprise three HVRs (HVR1, HVR2, HVR3).
[0042] A number of HVR delineations are in use and are encompassed herein. Exemplary HVRs for 4-chain antibodies and antigen-binding antibody fragments thereof herein include: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31 -35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) combinations of (a), (b), and/or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).
[0043] The amino acid residues of a single-domain antibody (such as VHH) can be numbered according to the general numbering for VH domains given by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, Md., Publication No. 91), as applied to VHH domains from Camelids in the article of Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240 (1-2): 185-195. According to this numbering, FR1 of a VHH comprises the amino acid residues at positions 1-30, CDR1 of a VHH comprises the amino acid residues at positions 31-35, FR2 of a VHH comprises the amino acids at positions 36-49, CDR2 of a VHH comprises the amino acid residues at positions 50-65, FR3 of a VHH comprises the amino acid residues at positions 66-94, CDR3 of a VHH comprises the amino acid residues at positions 95-102, and FR4 of a VHH comprises the amino acid residues at positions 103-113. In this respect, it should be noted that — as is well known in the art for VH domains and for VHH domains — the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering).
[0044] Unless otherwise indicated, CDR residues and other residues in the variable domain (e.g., framework, “FR,” residues) are numbered herein according to Kabat et al.
[0045] The term “cassette”, “expression cassette,” or “gene cassette” refers to a sequence of DNA carrying, and capable of directing the expression of, one or more genes of interest between one or more sets of restriction sites. It can be transferred from one DNA sequence (usually a vector) to another by “cutting” the fragment out using restriction enzymes and “pasting” it back into the new context (such as a viral genome). Typically, the DNA fragment (nucleic acid sequence) is operatively associated with expression control sequence elements which provide for the proper transcription and translation of the target nucleic acid sequence(s) (genes). Such sequence elements may include a promoter and a polyadenylation signal.
[0046] A sequence “encoding” an expression product, such as a polypeptide, is a minimum nucleotide sequence that, when expressed, results in the production of that polypeptide.
[0047] The term “exogenous” refers to a combination of elements not naturally occurring. For example, an “exogenous gene” refers to a gene to be introduced to the genome of a virus, wherein that gene is not normally found in the genome of the virus or is a homolog of a gene expressed in the virus from a different species (e.g., the bovine herpes virus UL49.5 gene, which encodes for a TAP inhibitor, is exogenous when inserted into a viral genome that does not natively encode UL49.5).
[0048] As used herein, the term “herpes simplex virus” or “HSV” refers to members of the Herpesviridae family. Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), also known by their taxonomical names Human alphaherpesvirus I and Human alphaherpesvirus 2, are two members of the human Herpesviridae family, a set of viruses that produce viral infections in the majority of humans.
[0049] “Percent (%) amino acid sequence identity” or “homology” with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
[0050] A coding sequence is “under the control of’ or “operatively associated with” a promoter in a virus or cell when RNA polymerase transcribes the coding sequence into RNA, particularly mRNA, which is then spliced (if it contains introns) and translated into the polypeptide encoded by the coding sequence.
[0051] The term “specific binding” or “specifically binds” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a KD for the target of at least about 10-4 M, alternatively at least about 10-5 M, alternatively at least about 10-6 M, alternatively at least about 10-7 M, alternatively at least about 10-8 M, alternatively at least about 10-9 M, alternatively at least about 10-10 M, alternatively at least about 10-11 M, alternatively at least about 10-12 M, or greater. In some embodiments, the term “specific binding” refers to binding where a molecule binds a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. KD can be determined by methods known in the art, such as ELISA, surface plasmon resonance (SPR), fluorescence activated cell sorting (FACS) analysis, or radioimmunoprecipitation (RIA). Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target.
[0052] An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non- human primates such as rhesus and cynomolgus monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.
[0053] A "cancer" refers herein to solid tumors.
[0054] "Treatment" or "therapy" of a subject refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of curing, reversing, alleviating, ameliorating, inhibiting, slowing down, or preventing the onset, progression, development, severity, or recurrence of a symptom, complication, condition, or biochemical indicia associated with a disease. In some embodiments, the disease is cancer.
[0055] An “effective amount” or "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems, or by assaying the activity of the agent in in vitro assays.
[0056] The phrase "pharmaceutically acceptable" indicates that the substance or composition must be compatible chemically and/or toxicologically, with the other ingredients comprising a formulation, and/or the mammal being treated therewith. [0057] As used herein, the term “no more than” refers to an amount that is less than or equal to. This may be an amount in integers. For example, no more than two lesions can refer to 0, 1, or 2 lesions.
[0058] As described herein, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Description of endpoints includes ranges between all endpoints disclosed. For example description of 1, 2, or 3 includes the ranges 1-2, 2-3 and 1-3.
[0059] II. Oncolytic viruses
[0060] Immunotherapy of cancer with oncolytic viruses is an emerging and maturing treatment modality which uses replication-competent viruses that selectively infect and damage tumor cells and may also, preferably, induce an immunological response which can control both the target tumor and distal tumors. Each species of oncolytic virus has a different cellular tropism, which helps determine which tissues are preferentially infected. Engineering of the virus can expand, restrict, or modulate this host range. A variety of species of virus have been investigated for use in oncolytic therapies, including those derived from HSV, vaccinia, and reovirus.
[0061] Thus, the present application provides oncolytic viruses that are effective for treating cancer. Non-limiting examples of oncolytic viruses include those derived from a herpes simplex virus, a vaccinia virus, an adenovirus, a reovirus, or a vesicular stomatitis virus. In some embodiments, the oncolytic virus (such as an oncolytic HSV) preferentially triggers an immune response that results in killing of tumor cells. As used herein, the virus “preferentially kills” tumor cells when certain infectious doses of the virus are more likely to kill tumor cells than neighboring healthy cells (such as at least two times more likely to kill tumor cells than neighboring healthy cells at a given dose). In some embodiments, the oncolytic virus expresses one or more payload proteins described below. In some embodiments, the oncolytic virus induces an immune response to the tumor, which, in some embodiments, causes tumor cells at sites distal to the site of infection to be killed. In some embodiments, the oncolytic virus is capable of evading an individual’s immune system after administration to the individual. As used herein, evading the individual’s immune system means that the oncolytic virus is able to preferentially replicate in tumor cells. In some embodiments, the oncolytic viruses provided herein are more sensitive to an innate antiviral response than a wild-type virus, enabling preferential replication in tumor cells. In some embodiments, the oncolytic viruses provided herein have an intermediate resistance to interferon.
III. Oncolytic herpes simplex virus
[0062] In one aspect, the present disclosure pertains to an oncolytic herpes simplex virus (HSV). In some embodiments, the oncolytic HSV is derived from HSV-1. In some embodiments the present disclosure pertains to a monotherapy comprising administering the HSV-1 virus intratumorally to an individual for treating solid tumors. In some embodiments the present disclosure pertains to a combination therapy comprising administering the HSV-1 virus intratumorally and administering an antibody that binds to PD-1 to an individual for treating solid tumors. In some embodiments, the oncolytic HSV comprises one or more expression cassettes described herein. In some embodiments, the oncolytic HSV expresses one or more payload proteins described herein. In some embodiments, the oncolytic HSV lacks one or more native HSV genes. In some embodiments, the oncolytic HSV lacks one or both copies of γ34.5. In some embodiments, the oncolytic HSV does not express one or more native HSV proteins, such as US 12. In some embodiments, the oncolytic HSV expresses one or more additional copies of a native HSV protein, such as US11. In some embodiments, the oncolytic HSV expresses a native HSV protein in a different temporal order, such as expressing immediate-early US11. The oncolytic HSV may be a component of a pharmaceutical composition described herein. The oncolytic HSV, or a pharmaceutical composition comprising the oncolytic HSV, may be administered to individual according to the methods described herein (such as the methods of treatment described herein). In some embodiments, the oncolytic HSV preferentially triggers an immune response that results in killing of tumor cells compared to the wild-type HSV from which it is derived. In some embodiments, the oncolytic HSV is capable of triggering an immune response that triggers killing tumor cells at one or more sites distal to a target site.
IV. Viral payloads
[0063] In some embodiments, the oncolytic virus (such as an oncolytic HSV), or the gene cassette otherwise described herein, comprises one or more genes encoding one or more payload molecules. The payload molecules are generally intended to enhance the therapeutic effectiveness of the oncolytic virus (such as an oncolytic HSV). For example, a payload molecule may promote an immune response (e.g., against the tumor target) or may enhance the cytotoxicity of the oncolytic virus.
IV- A. IL- 12
[0064] In some embodiments, an oncolytic virus (such as an oncolytic HSV), or an expression cassette otherwise described herein, comprises a polynucleotide encoding interleukin 12 (IL- 12). [0065] IL-12 is a heterodimeric protein comprising two subunits: p35 and p40. The native p35 subunit is linked to the p40 subunit by a disulfide bond. The human and mouse p40 subunits are 70% identical, while the p35 subunits share 60% amino acid sequence homology. The p35 and p40 subunits may function in receptor binding and signal transduction, respectively (Zou, J. J., et al. (1995). Structure-function analysis of the p35 subunit of mouse interleukin 12. The Journal of biological chemistry, 270(11), 5864-5871). IL-12 is normally secreted by antigen-presenting cells, such as macrophages and dendritic cells. Biologically active IL- 12 (comprising both subunits in a heterodimer) functions to differentiate naive T cells into Th1 cells, promote cytotoxic activity of NK cells and T cells, and block angiogenesis.
[0066] In some embodiments, the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises a polynucleotide encoding the p35 subunit of IL- 12 and/or a polynucleotide encoding the p40 subunit of IL- 12. In some embodiments, the p35 subunit and/or p40 subunit of IL-12 is human. In some embodiments, the p35 subunit and/or p40 subunit of IL- 12 is murine. In some embodiments, the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises a polynucleotide encoding an IL- 12 heterodimer comprising a p35 subunit and a p40 subunit. In some embodiments, the IL- 12 heterodimer comprises a polypeptide comprising a p35 subunit of IL- 12 and a p40 subunit of IL- 12 connected by a peptide linker.
[0067] In some embodiments, the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises a polynucleotide encoding a human p35 subunit of IL- 12 and/or a polynucleotide encoding a human p40 subunit of IL- 12. In some embodiments, the human p35 subunit comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the human p40 subunit comprises the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the human p40 subunit comprises the amino acid sequence of SEQ ID NO:9, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises a polynucleotide encoding an IL- 12 heterodimer comprising a human p35 subunit and a human p40 subunit. In some embodiments, the IL- 12 heterodimer comprises a polypeptide comprising a human p35 subunit of IL- 12 and a human p40 subunit of IL- 12 connected by a peptide linker. In some embodiments, the peptide linker comprises an amino acid sequence comprising glycine and serine residues. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the IL-12 heterodimer comprises the amino acid sequence of SEQ ID NO:4In some embodiments, the IL-12 heterodimer comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 10. [0068] In some embodiments, the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises a polynucleotide encoding a murine p35 subunit of IL-12 and/or a polynucleotide encoding a murine p40 subunit of IL-12. In some embodiments, the murine p35 subunit comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about
88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about
93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about
98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the murine p40 subunit comprises the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about
87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about
92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about
97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in
SEQ ID NO: 6. In some embodiments, the murine p40 subunit comprises the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having any of at least about 80%, at least about
81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about
86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about
91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about
96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises a polynucleotide encoding an IL- 12 heterodimer comprising a murine p35 subunit and a murine p40 subunit. In some embodiments, the IL- 12 heterodimer comprises a polypeptide comprising a murine p35 subunit of IL- 12 and a murine p40 subunit of IL- 12 connected by a peptide linker. In some embodiments, the peptide linker comprises an amino acid sequence comprising glycine and serine residues. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID N0:7In some embodiments, the IL-12 heterodimer comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 12.
IV-B. CD40 agonist
[0069] Cluster of differentiation 40 (CD40) is a costimulatory polypeptide expressed on numerous cell types, from antigen presenting cells (APCs) to epithelial cells. It is additionally present on various cancer cells. CD40 agonist, also known as cluster of differentiation 154 (CD154), comprises 261 amino acids and is a type II membrane glycopolypeptide that is expressed on the surface of activated T cells. Native CD40 agonist promotes B cell maturation. It is additionally essential for immunoglobulin class switching, as lack of CD40 agonist is associated with hyper IgM syndrome. CD40 agonist exists as a membrane-bound form, in which the extracellular domain forms a homotrimer, and a proteolytically-cleaved, soluble form, which has been shown to be biologically active.
[0070] In some embodiments, provided herein is an oncolytic virus comprising a polynucleotide encoding a CD40 agonist. In some embodiments, an oncolytic virus (such as an oncolytic HSV), or an expression cassette otherwise described herein, comprises a polynucleotide encoding CD40 agonist. In some embodiments, the CD40 agonist is a CD40 ligand. In some embodiments, the CD40 agonist comprises a CD40 ligand ectodomain. In some embodiments, the CD40 agonist is a trimer of three single-chain trimeric CD40 ligand ectodomains. In some embodiments, each of the three single-chain trimeric CD40 ligand ectodomains is fused to a trimerization motif, e.g., to direct formation of the trimer of three single-chain trimeric CD40 ligand ectodomains. In some embodiments, each of the three single-chain trimeric CD40 ligand ectodomains is fused to an Fc region, e.g., to direct formation of the trimer of three single-chain trimeric CD40 ligand ectodomains. In some embodiments, said Fc region is an IgG Fc region e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region. In some embodiments, said Fc region comprises one or more amino acid substitutions, insertions, or deletions that disfavor binding of said Fc region to another Fc region, such as an IgG Fc region, e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region. In some embodiments, said Fc region comprises a substitution of the IgG interaction domain with an IgA interaction domain. In some embodiments, each of the three single-chain trimeric CD40 ligand ectodomains is bivalent. In some embodiments, the CD40 agonist is an agonist antibody.
[0071] In some embodiments, the CD40 agonist comprises a human CD40 ligand ectodomain. In some embodiments, the human CD40 ligand ectodomain comprises the amino acid sequence set forth in SEQ ID NO:20, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the CD40 agonist is a trimer of three single-chain trimeric human CD40 ligand ectodomains. In some embodiments, the single-chain trimeric human CD40 ligand ectodomains comprise a polypeptide comprising three human CD40 ligand ectodomains connected by peptide linkers. In some embodiments, the single-chain trimeric human CD40 ligand ectodomain polypeptide comprises a first human CD40 ligand ectodomain connected by a peptide linker to a second human CD40 ligand ectodomain which is connected by a peptide linker to a third human CD40 ligand ectodomain. In some embodiments, the peptide linker comprises glycine and serine residues. In some embodiments, the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, the CD40 agonist comprises a trimerization motif operably linked to each of the three single-chain trimeric CD40 ligand ectodomains. In some embodiments, the trimerization motif is a T4 fibritin trimerization motif. In some embodiments, the T4 fibritin trimerization motif comprises the amino acid sequence set forth in SEQ ID NO:21. In some embodiments, the trimerization motif is linked to each of the three single-chain trimeric CD40 ligand ectodomains by a peptide linker. In some embodiments, the peptide linker connecting the trimerization motif to each of the three single-chain trimeric CD40 ligand ectodomains comprises an amino acid sequence comprising of glycine and/or serine residues. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO:23In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO:27. In some embodiments, the CD40 agonist further comprises a signal peptide sequence operably linked to each of the three single-chain trimeric CD40 ligand ectodomains. In some embodiments, the signal peptide sequence comprises the amino acid sequence of SEQ ID NO:24. [0072] In some embodiments, the CD40 agonist comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:25, or an amino acid sequence having any of about at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:25. In some embodiments, the CD40 agonist comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having any of about at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:30. In some embodiments, the CD40 agonist forms or is a trimer comprising three polypeptides comprising the amino acid sequence of SEQ ID NO:25, or an amino acid sequence having any of about at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:25. In some embodiments, the CD40 agonist forms or is a trimer comprising three polypeptides comprising the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having any of about at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:30. [0073] In some embodiments, the CD40 agonist comprises a murine CD40 ligand ectodomain. In some embodiments, the murine CD40 ligand ectodomain comprises the amino acid sequence set forth in SEQ ID NO:26, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the CD40 agonist is a trimer of three single-chain trimeric murine CD40 ligand ectodomains. In some embodiments, the single-chain trimeric murine CD40 ligand ectodomains comprise a polypeptide comprising three murine CD40 ligand ectodomains connected by peptide linkers. In some embodiments, the single-chain trimeric murine CD40 ligand ectodomain polypeptide comprises a first murine CD40 ligand ectodomain connected by a peptide linker to a second murine CD40 ligand ectodomain which is connected by a peptide linker to a third murine CD40 ligand ectodomain. In some embodiments, the peptide linker comprises glycine and serine residues. In some embodiments, the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, the CD40 agonist comprises a trimerization motif operably linked to each of the three single-chain trimeric CD40 ligand ectodomains. In some embodiments, the trimerization motif is a T4 fibritin trimerization motif. In some embodiments, the T4 fibritin trimerization motif comprises the amino acid sequence set forth in SEQ ID NO:21. In some embodiments, the trimerization motif is linked to each of the three single-chain trimeric CD40 ligand ectodomains by a peptide linker. In some embodiments, the peptide linker connecting the trimerization motif to each of the three single-chain trimeric CD40 ligand ectodomains comprises an amino acid sequence comprising of leucine, glycine, and/or serine residues. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO:23. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO:27. In some embodiments, the CD40 agonist further comprises a signal peptide sequence operably linked to each of the three single-chain trimeric CD40 ligand ectodomains. In some embodiments, the signal peptide sequence comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, the CD40 agonist comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 28 In some embodiments, the CD40 agonist comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:29, or an amino acid sequence having any of about at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:29. In some embodiments, the CD40 agonist forms a trimer comprising three polypeptides comprising the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:29.
IV-C. CTLA-4 binding protein
[0074] Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4 or CTLA-4), also known as cluster of differentiation 152 (CD 152), is a polypeptide receptor that functions as an immune checkpoint and downregulates immune responses. The polypeptide contains an extracellular V-like domain, a transmembrane domain, and a cytoplasmic tail. Alternate isoforms have been characterized. CTLA-4 is constitutively expressed in regulatory T cells, but is only upregulated in conventional T cells after activation, and contributes to the inhibitory function of regulatory T cells. CTLA-4 binds to CD80 and CD86, also known as B7-1 and B7-2 respectively, on APCs in order to induce its inhibitory function to T cells.
[0075] In some embodiments, an oncolytic virus (such as an oncolytic HSV), or an expression cassette otherwise described herein, comprises a polynucleotide encoding a CTLA-4 binding protein. In some embodiments, the CTLA-4 binding protein is a CTLA-4 antagonist. Lor example, in some instances, the CTLA-4 binding protein inhibits the interaction between CTLA-4 and one or more CTLA-4 ligands, such as CD80 and/or CD86. In some embodiments, the CTLA-4 binding protein specifically binds to human CTLA-4, murine CTLA-4, or both human and murine CTLA- 4.
[0076] In some embodiments, the CTLA-4 binding protein is an anti-CTLA-4 antibody or antigen binding fragment thereof. In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof specifically binds to human CTLA-4, murine CTLA-4, or both human and murine CTLA-4. In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment is bivalent. In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment comprises an Fc region, such as an active Fc region. In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment comprises an IgG1, IgG2, IgG3, or IgG4 constant domain, e.g., a human or mouse IgG1, IgG2, IgG3, or IgG4 constant domain. In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof comprises a single-chain variable fragment (scFv). In some embodiments, the anti-CTLA-4 scFv is fused to the N-terminus of an IgG1, IgG2, IgG3, or IgG4 constant domain, e.g., a human or mouse IgG1, IgG2, IgG3, or IgG4 constant domain. In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof comprises an anti-CTLA-4 VHH, e.g., a camelid antibody comprising an anti-CTLA-4 VHH. In some embodiments, the anti-CTLA-4 VHH is fused to the heavy chain of an IgG1, IgG2, IgG3, or IgG4 Fc, e.g., a human or mouse IgG1, IgG2, IgG3, or IgG4 Fc.
[0077] In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof, such as the anti-CTLA-4 scFv, specifically binds to human CTLA-4. In some embodiments, the anti- CTLA-4 scFv is fused to the N-terminus of a IgG1 constant domain, e.g., a human IgG1 constant domain. In some embodiments, the human IgG1 is a variant human IgG1 comprising a C220S substitution, wherein the numbering of the residues is according to EU numbering. In some embodiments, the human IgG1 is a G1m(17) IgG1. In some embodiments, the anti-CTLA-4 antibody causes depletion of regulatory T (Treg) cells.
[0078] In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti-CTLA-4 scFv) comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises one or more of: (a) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:40; (b) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO:41; and (c) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:42; and/or wherein the VL comprises one or more of: (a) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO:43; (b) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO:44; and (c) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO:45.
[0079] In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti-CTLA-4 scFv) comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:46, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:46; and/or a VL comprising the amino acid sequence set forth in SEQ ID NO:47, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:47. In some embodiments, the variable heavy chain and variable light chain are connected via a linker sequence. In some embodiments, the linker sequence comprises an amino acid sequence set forth in SEQ ID NO:61.
[0080] In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti-CTLA-4 scFv) comprises an IgG1 constant domain comprising the amino acid sequence set forth in SEQ ID NO:48, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:48. In some embodiments, the heavy chain of the CTLA- 4 antibody comprises the amino acid sequence set forth in SEQ ID NO48, with or without the C terminal lysine.
[0100] In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti-CTLA-4 scFv) comprises the amino acid sequence set forth in SEQ ID NO:60, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:60.
[0101] In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti-CTLA-4 scFv) comprises a signal peptide sequence comprising the amino acid sequence set forth in SEQ ID NO:49. In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti-CTLA-4 scFv) comprises the amino acid sequence set forth in SEQ ID NO: 50, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 50.
[0102] In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof, such as the anti-CTLA-4 VHH, specifically binds to murine CTLA-4. In some embodiments, the anti- CTLA-4 VHH is fused to the heavy chain of a murine IgG2a Fc.
[0081] In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti-CTLA-4 VHH) comprises a variable heavy chain (VH), wherein the VH comprises one or more of: (a) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:51; (b) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 52; and (c) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 53. In some embodiments, the anti- CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti-CTLA-4 VHH) comprises a VH, wherein the VH comprises a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 51 , a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 52, and a CDR- H3 comprising the amino acid sequence set forth in SEQ ID NO: 53. In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti-CTLA-4 VHH) comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 54, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 54. In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti- CTLA-4 VHH) comprises the amino acid sequence set forth in SEQ ID NO: 58, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 58. In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti- CTLA-4 VHH) comprises the amino acid sequence set forth in SEQ ID NO: 59, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about
83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about
88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about
93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about
98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 59.
[0103] In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti-CTLA-4 VHH) comprises a signal peptide sequence comprising the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti-CTLA-4 VHH) comprises the amino acid sequence set forth in SEQ ID NO: 56. In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti-CTLA-4 VHH) comprises the amino acid sequence set forth in SEQ ID NO:57.
[0104] In some embodiments, the oncolytic herpes simplex type 1 (HSV-1) virus comprising a polynucleotide encoding an antibody to human cytotoxic T lymphocyte-associated protein 4 (αCTLA4) is administered to an individual, and an antibody to human cytotoxic T lymphocyte- associated protein 4 (αCTLA4) can further be administered to the individual. In some embodiments, the antibody to human cytotoxic T lymphocyte-associated protein 4 (αCTLA4) encoded within the oncolytic virus and antibody to human cytotoxic T lymphocyte-associated protein 4 (αCTLA4) administered systemically to the individual are the same antibody. In some embodiments, the antibody to human cytotoxic T lymphocyte-associated protein 4 (αCTLA4) encoded within the oncolytic virus and antibody to human cytotoxic T lymphocyte-associated protein 4 (αCTLA4) administered systemically to the individual are the different antibodies.
IV-D. FLT3 ligand
[0105] In some embodiments, an oncolytic virus (such as an oncolytic HSV), or an expression cassette otherwise described herein, comprises a polynucleotide encoding a fms-like tyrosine kinase 3 ( FLT3) ligand (FLT3L). FLT3L is a growth and differentiation factor that enhances and expands dendritic cells (DCs) as well as recruits DCs to the tumor microenvironment. [0106] In some embodiments, the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises a polynucleotide encoding a human FLT3L. In some embodiments, the human FLT3L comprises the amino acid sequence of SEQ ID NO: 72, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 72.
[0107] In some embodiments, the human FLT3L comprises a signal peptide directing secretion to the plasma membrane. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 70. In some embodiments, the human FLT3L comprises the amino acid sequence of SEQ ID NO:71, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 71.
[0108] In some embodiments, the FLT3L, e.g., the human FLT3L, is a homodimer. In some embodiments, the human FLT3L is proteolytically processed into soluble FLT3L. In some embodiments, the soluble FLT3L forms a homodimer.
IV-E. Other pay load molecules
[0109] In some embodiments, an oncolytic virus (such as an oncolytic HSV), or an expression cassette otherwise described herein, comprises one or more polynucleotides encoding a US 11 protein, such as a US11 protein from an HSV, e.g., an HSV-1 or HSV-2.
[0110] The protein kinase R (PKR) pathway is a component of the host cellular innate anti-viral response. PKR becomes activated in response to binding double-stranded RNA (dsRNA), a byproduct of viral replication, leading to phosphorylation and inactivation of eukaryotic translation initiation Factor 2 Subunit 1 (eIF2α), a translation initiation factor. Phosphorylated eIF2α prevents translation initiation, a cellular defense mechanism aimed at blocking the production of viral proteins. The US11 protein is believed to bind and sequester dsRNA, preventing the activation of the PKR pathway in host cells, and enabling enhanced viral replication.
[0111] In some embodiments, the US 11 protein comprises the amino acid sequence of SEQ ID NO:80, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 80.
[0112] In some embodiments, the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises a polynucleotide encoding a US11 protein, wherein the polynucleotide comprises a native US11 gene nucleotide sequence, e.g., from an HSV, such as an HSV-1 or an HSV-2. In some embodiments, the native US11 gene is a native US11 late gene, wherein the US11 protein is expressed in the late stage of viral replication. In some embodiments, the native US11 late gene is under the control of the endogenous US11 promoter, e.g., from an HSV, such as an HSV-1 or an HSV-2.
[0113] In some embodiments, the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises a polynucleotide comprising a variant US11 gene. In some embodiments, the variant US 11 gene is codon optimized for expression of the US 11 protein in human cells. In some embodiments, the variant US11 gene encodes a wild type US11 protein, e.g., from an HSV, such as an HSV-1 or an HSV-2. In some embodiments, the variant US11 gene comprises the nucleotide sequence of SEQ ID NO:204, or a nucleotide sequence having any of at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the nucleotide sequence of SEQ ID NO:204. In some embodiments, the variant US 11 gene is operably linked to a promoter. In some embodiments, the promoter directs immediate early expression of the US 11 protein during viral replication. In some embodiments, the promoter is an endogenous US 12 promoter from an HSV, such as HSV-1 or HSV-2, or a portion thereof.
[0114] In some embodiments, the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises both a polynucleotide encoding a US11 protein and comprising a native US11 gene nucleotide sequence, e.g., as described above; and a polynucleotide comprising a variant US11 gene, e.g., as described above.
[0115] In some embodiments, an oncolytic virus (such as an oncolytic HSV), or an expression cassette otherwise described herein, comprises a polynucleotide encoding a transporter associated with antigen processing (TAP) inhibitor, such as a viral TAP inhibitor. In general, viral TAP inhibitors prevent TAP from transporting peptides into the lumen of the endoplasmic reticulum, thus impairing peptide loading onto major histocompatibility complex (MHC) Class I molecules for display at the cell surface (Verweij et al. Viral inhibition of the transporter associated with antigen processing (TAP): A striking example of functional convergent evolution. PLoS Pathog. 2015; 11(4): el 004743). Although TAP inhibition disrupts the transport of newly-expressed MHC molecules to the cell surface, this does not block pre-existing antigen display. Thus, TAP inhibition by a TAP inhibitor can prevent the display of viral antigens on the cell surface, preventing premature clearance of infected cells and enabling virus persistence throughout multiple rounds of virus replication.
[0116] In some embodiments, the TAP inhibitor is derived from herpes virus 1 or herpes virus 2. In some embodiments, the TAP inhibitor is derived from bovine herpes virus 1. In some embodiments, the TAP inhibitor is any of UL49.5, US6, or ICP47. In some embodiments, the TAP inhibitor is UL49.5. In some embodiments, the TAP inhibitor comprises the amino acid sequence of SEQ ID NO:83, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the TAP inhibitor further comprises a signal peptide sequence. In some embodiments, the signal peptide sequence comprises the amino acid sequence of SEQ ID NO: 81. In some embodiments, the TAP inhibitor comprises the amino acid sequence of SEQ ID NO: 82, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 82. In some embodiments, the TAP inhibitor is expressed during the immediate early phase of viral replication, i.e., it is expressed as an immediate early gene. In some embodiments, the polynucleotide encoding the TAP inhibitor is expressed under the control of an immediate early promoter, such as a CMV promoter, e.g., an hCMV promoter.
V. Expression cassettes
[0117] Provided herein are one or more expression cassettes comprising a polynucleotide encoding IL- 12, a polynucleotide encoding a CD40 agonist, a polynucleotide encoding a CTLA-4 binding protein, a polynucleotide encoding an FLT3 ligand (FLT3L), or any combination thereof.
V-A. Expression cassettes encoding IL- 12, a CD40 agonist, and/or a CTLA-4 binding protein
[0118] Provided herein are expression cassettes comprising a polynucleotide encoding IL- 12, a polynucleotide encoding a CD40 agonist, and/or a polynucleotide encoding a CTLA-4 binding protein.
[0119] In some embodiments, the expression cassettes of the disclosure comprise a promoter operably linked to each of the polynucleotide encoding IL- 12, the polynucleotide encoding the CD40 agonist, and/or the polynucleotide encoding the CTLA-4 binding protein. Any suitable promoter may be used in the cassettes of the disclosure, so long as the promoter drives expression of the associated polynucleotide. Exemplary and non-limiting promoters that may be used include the human cytomegalovirus (hCMV) promoter, the murine cytomegalovirus (mCMV) promoter, the Aotine betaherpesvirus 1 (AoHV 1) promoter, the CAG promoter, a CMV hybrid promoter, the EF1a promoter, the MMLV 5’ long terminal repeat (LTR) from the Moloney murine leukemia virus promoter (i.e., the MMLV promoter), the Pbidir3 promoter, and a native HSV promoter sequence, such as the HSV-1 or HSV-2 US 12 promoter, or the HSV-1 or HSV-2 US11 promoter. [0120] In some embodiments, the expression cassettes of the disclosure comprise a polyadenylation signal operably linked to each of the polynucleotide encoding IL- 12, the polynucleotide encoding the CD40 agonist, and/or the polynucleotide encoding the CTLA-4 binding protein. Any suitable polyadenylation signal may be used in the cassettes of the disclosure. Exemplary and non-limiting polyadenylation signals (polyA or pA) that may be used include the simian vacuolating virus 40 polyA (SV40pA), the human beta globin polyA (hBGpA), the human growth hormone polyA(hGH polyA), the rabbit beta globin polyA (rBGpA), a bovine growth hormone polyadenylation (BGHpA), a polyA derived from the human GAPDH gene, and a native HSV polyA sequence, such as the US10-12 polyA or the US9-10 polyA from HSV-1 or HSV-2. [0121] In some embodiments, the expression cassettes of the disclosure may comprise any suitable promoter and/or polyadenylation signal known in the art or described herein operably linked to any of the polynucleotide encoding IL- 12, the polynucleotide encoding the CD40 agonist, and/or the polynucleotide encoding the CTLA-4 binding protein.
[0122] In some embodiments, the expression cassettes of the disclosure comprise an RNA Polymerase II transcriptional pause signal positioned after each of the polynucleotide encoding IL- 12, the polynucleotide encoding the CD40 agonist, and/or the polynucleotide encoding the CTLA-4 binding protein. Any suitable RNA Polymerase II transcriptional pause signal may be used in the cassettes of the disclosure. Exemplary and non-limiting RNA polymerase II transcriptional pause signals include the human complement C2 protein terminator (C2) and the human Gastrin terminator (hGT).
[0123] In some embodiments, an expression cassette of the disclosure comprises, in order, the polynucleotide encoding the CTLA-4 binding protein, the polynucleotide encoding the CD40 agonist, and the polynucleotide encoding the IL- 12. In some embodiments, the polynucleotide encoding the CTLA-4 binding protein and the polynucleotide encoding the IL- 12 are in the same orientation in the expression cassette, and the polynucleotide encoding the CD40 agonist is in the reverse orientation relative to the polynucleotide encoding the CTLA-4 binding protein and the polynucleotide encoding the IL- 12.
[0124] In some embodiments, the polynucleotide encoding the CTLA-4 binding protein is operably linked to a promoter, such as any suitable promoter known in the art or described herein. In some embodiments, the promoter is an mCMV promoter. In some embodiments, the expression cassette comprises a polyadenylation signal operably linked to the polynucleotide encoding the CTLA-4 binding protein, such as any suitable polyadenylation signal known in the art or described herein. In some embodiments, the polyadenylation signal is a polyA derived from the human GAPDH gene.. In some embodiments, the expression cassette further comprises a Kozak sequence positioned between the promoter and the polynucleotide encoding the CTLA-4 binding protein In some embodiments, the expression cassette further comprises an RNA polymerase II transcriptional pause signal positioned after the polyadenylation signal, such as any suitable RNA polymerase II transcriptional pause signal known in the art or described herein. In some embodiments, the RNA polymerase II transcriptional pause signal is a C2 RNA polymerase II transcriptional pause signal. In some embodiments, the encoded CTLA-4 binding protein is any of the CTLA-4 binding proteins described herein, e.g., in Section III-C, above.
[0125] In some embodiments, the polynucleotide encoding the CD40 agonist is operably linked to a promoter, such as any suitable promoter known in the art or described herein. In some embodiments, the promoter is the AOHV1 promoter. In some embodiments, the expression cassette further comprises a polyadenylation signal operably linked to the polynucleotide encoding the CD40 agonist, such as any suitable polyadenylation signal known in the art or described herein. In some embodiments, the polyadenylation signal is a hBGpA. In some embodiments, the expression cassette further comprises a Kozak sequence positioned between the promoter and the polynucleotide encoding the CD40 agonist. In some embodiments, the expression cassette further comprises an RNA polymerase II transcriptional pause signal positioned after the polyadenylation signal, such as any suitable RNA polymerase II transcriptional pause signal known in the art or described herein. In some embodiments, the RNA polymerase II transcriptional pause signal is the hGT RNA polymerase II transcriptional pause signal. In some embodiments, the encoded CD40 agonist is any of the CD40 agonists described herein, e.g., in Sections III-B or V, herein. In some embodiments, the polynucleotide encoding the CD40 agonist is in the reverse orientation within the expression cassette relative to the polynucleotide encoding the IL- 12 and the polynucleotide encoding the CTLA-4 binding protein.
[0126] In some embodiments, the polynucleotide encoding IL- 12 is operably linked to a promoter, such as any suitable promoter known in the art or described herein. In some embodiments, the promoter is the MMLV promoter In some embodiments, the expression cassette further comprises a polyadenylation signal positioned after the polynucleotide encoding IL- 12, such as any suitable polyadenylation signal known in the art or described herein. In some embodiments, the polyadenylation signal is the US 10- 12 poly A or the US 9- 10 polyA from HSV, such as from HSV- 1 or HSV-2. In some embodiments, the US 10- 12 polyA comprises the nucleotide sequence of a native HSV-1 or HSV-2 US 10- 12 poly A. In some embodiments, the expression cassette further comprises a Kozak sequence positioned between the promoter and the polynucleotide encoding IL- 12.
[0127] In some embodiments, an expression cassette of the disclosure further comprises a polynucleotide encoding a US 10 protein and/or a polynucleotide encoding a US 11 protein; or a polynucleotide encoding a US11 protein and a US 10 protein.
[0128] In some embodiments, an expression cassette of the disclosure comprises a polynucleotide encoding a US 10 protein and/or a polynucleotide encoding a US11 protein. In some embodiments, the polynucleotide encoding the US 11 protein is operably linked to a promoter, such as any suitable promoter known in the art or described herein. In some embodiments, the promoter is an endogenous US11 promoter from an HSV, such as HSV-1 or HSV-2. In some embodiments, the endogenous US11 promoter directs late expression of the US11 protein during viral replication. In some embodiments, the polynucleotide encoding the US 10 protein is operably linked to a promoter, such as any suitable promoter known in the art or described herein. In some embodiments, the promoter is an endogenous US 10 promoter. In some embodiments, the expression cassette comprises a polyadenylation signal operably linked to the polynucleotide encoding the US 10 protein, such as any suitable polyadenylation signal known in the art or described herein. In some embodiments, the polyadenylation signal is a hGHpolyA. In some embodiments, the encoded US 11 protein is an HSV US 11 protein, such as an HSV-1 or HSV-2 US 11 protein. In some embodiments, the polynucleotide encoding the US 11 protein comprises a native US 11 gene. In some embodiments, the expression cassette comprises, in order, the polynucleotide encoding the US11 protein (e.g., comprising a native US11 gene) and/or the polynucleotide encoding the US 10 protein, the polynucleotide encoding the CTLA-4 binding protein, the polynucleotide that encodes the CD40 agonist, and the polynucleotide encoding the IL- 12. In some embodiments, the polynucleotides encoding the CTLA-4 binding protein, the IL- 12, the US 11 protein and/or the US 10 protein are in the same orientation in the expression cassette, and the polynucleotide that encodes the CD40 agonist is in the reverse orientation relative to the polynucleotides encoding the CTLA-4 binding protein, the IL- 12, the US 11 protein and/or the US 10 protein.
[0129] In some embodiments, an expression cassette of the disclosure comprises a polynucleotide encoding a US11 protein and a US 10 protein. In some embodiments, the polynucleotide encoding the US 11 protein and the US 10 protein comprises a nucleic acid sequence encoding the US 11 protein, and a nucleic acid sequence encoding the US 10 protein. In some embodiments, at least a portion of the nucleic acid sequence encoding the US11 protein overlaps with at least a portion of the nucleic acid sequence encoding the US 10 protein. In some embodiments, the nucleic acid sequence encoding the US 11 protein is operably linked to a promoter, such as any suitable promoter known in the art or described herein. In some embodiments, the promoter is an endogenous US11 promoter from an HSV, such as HSV-1 or HSV-2. In some embodiments, the endogenous US11 promoter directs late expression of the US11 protein during viral replication. In some embodiments, the nucleic acid sequence encoding the US 10 protein is operably linked to a promoter. In some embodiments, the promoter is a native US 10 promoter from an HSV, such as HSV-1 or HSV-2. In some embodiments, the promoter is embedded within the nucleic acid sequence encoding the US11 protein. In some embodiments, the encoded US11 protein is an HSV US 11 protein, such as an HSV-1 or HSV-2 US 11 protein. In some embodiments, the expression cassette comprises a polyadenylation signal operably linked to the nucleic acid sequence encoding the US 10 protein, such as any suitable polyadenylation signal known in the art or described herein. In some embodiments, the polyadenylation signal is a hGHpolyA. In some embodiments, the polynucleotide encoding the US 11 protein and the US 10 protein comprises a native US 11 gene. In some embodiments, the expression cassette comprises, in order, the polynucleotide encoding the US11 protein and the US 10 protein (e.g., comprising a native US11 gene), the polynucleotide encoding the CTLA-4 binding protein, the polynucleotide that encodes the CD40 agonist, and the polynucleotide encoding the IL- 12. In some embodiments, the polynucleotides encoding the CTLA-4 binding protein, the IL- 12, and the US 11 and US 10 proteins are in the same orientation in the expression cassette, and the polynucleotide that encodes the CD40 agonist is in the reverse orientation relative to the polynucleotides encoding the CTLA-4 binding protein, the IL- 12, and the US11 and US 10 proteins.
[0130] In some embodiments, an expression cassette of the disclosure further comprises a polynucleotide encoding a US 11 protein, wherein the polynucleotide comprises a variant US 11 gene. In some embodiments, the variant US11 gene comprises a sequence that is codon optimized for expression of the US11 protein in human cells. In some embodiments, the variant US11 gene is operably linked to a promoter, such as any suitable promoter known in the art or described herein. In some embodiments, the promoter is an endogenous US 12 promoter from an HSV, such as HSV-1 or HSV-2, or a portion thereof. In some embodiments, the endogenous US 12 promoter, or the portion thereof, directs immediate early expression of the US 11 protein during viral replication. In some embodiments, the expression cassette further comprises a 5’ untranslated region (UTR) sequence positioned between the promoter and the variant US 11 gene.. In some embodiments, the expression cassette further comprises a polynucleotide encoding a US 12 protein positioned after the variant US11 gene (e.g., after a stop codon in the variant US11 gene). In some embodiments, the US12 protein is from an HSV, such as HSV-1 or HSV-2. In some embodiments, the polynucleotide encoding the US 12 protein is not operably linked to a promoter. In some embodiments, the encoded US 12 protein is not expressed. In some embodiments, the expression cassette further comprises a spacer sequence and a UTR sequence positioned between the variant US 11 gene and the polynucleotide encoding the US 12 protein. In some embodiments, the expression cassette comprises, in order, the variant US11 gene; the polynucleotides encoding the US 10 and/or US 11 proteins, or the polynucleotide encoding the US 10 and US 11 proteins; the polynucleotide encoding the CTLA-4 binding protein; the polynucleotide that encodes the CD40 agonist; and the polynucleotide encoding the IL- 12. In some embodiments, the polynucleotides encoding the CTLA-4 binding protein, the IL- 12, and the US 10 and/or US11 proteins are in the same orientation in the expression cassette, and the polynucleotide that encodes the CD40 agonist is in the reverse orientation relative to the polynucleotides encoding the CTLA-4 binding protein, the IL- 12, and the US 10 and/or US11 proteins.
[0131] In some embodiments, an expression cassette of the disclosure comprises, in order, a promoter (e.g., an HSV US 12 promoter) operably linked to the polynucleotide comprising a variant US 11 gene; optionally, a 5’ UTR sequence; the polynucleotide comprising the variant US 11 gene; a promoter (e.g., a native HSV US 11 promoter); the polynucleotide encoding the US 11 protein and the US10 protein; a polyadenylation signal (e.g., a hGHpA poly A) operably linked to the polynucleotide encoding the US11 protein and the US10 protein; a promoter (e.g., a CMV promoter such as an mCMV promoter) that directs expression of the polynucleotide encoding the CTLA-4 binding protein; optionally, a Kozak sequence for expression of the polynucleotide encoding the CTLA-4 binding protein; the polynucleotide encoding the CTLA-4 binding protein; a polyadenylation signal (e.g., a GAPDH SpA poly A) that is operably linked to the polynucleotide encoding the CTLA-4 binding protein; optionally, an RNA polymerase II pause site (e.g., a C2 pause site); an RNA polymerase II pause site (e.g., an hGT pause site); a polyadenylation signal (e.g., an hBGpA poly A) that is operably linked to the polynucleotide encoding the CD40 agonist; the polynucleotide that encodes the CD40 agonist; optionally, a Kozak sequence for expression of the polynucleotide that encodes the CD40 agonist; a promoter (e.g., an AoHVl promoter) that controls expression of the CD40 agonist; a promoter (e.g., an MMLV promoter) that controls expression of the IL- 12; optionally, a Kozak sequence for expression of the polynucleotide encoding the IL- 12; the polynucleotide encoding the IL- 12; and a polyadenylation signal (e.g., an HSV US 10- 12 poly A) that is operably linked to the polynucleotide encoding the IL- 12.
[0132] In some embodiments, an expression cassette of the disclosure comprises the nucleotide sequence of SEQ ID NO:201, or a nucleotide sequence having any of at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the nucleotide sequence of SEQ ID NO:201. In some embodiments, an expression cassette of the disclosure comprises the nucleotide sequence of SEQ ID NO:202, or a nucleotide sequence having any of at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the nucleotide sequence of SEQ ID NO:202.
[0133] In some embodiments, an expression cassette of the disclosure is integrated into a genome of a virus, such as an oncolytic HSV, e.g., an oncolytic HSV-1 or oncolytic HSV-2. In some embodiments, the cassette is integrated in the US10-12 locus of an oncolytic HSV, e.g., an oncolytic HSV-1 or oncolytic HSV-2.
[0134] In some embodiments, the expression cassette comprises: (i) the polynucleotide encoding IL- 12, (ii) the polynucleotide encoding the CD40 agonist, (iii) and the polynucleotide encoding the CTLA-4 binding protein, e.g., as described above. In some embodiments, the expression cassete is in the orientation relative to the internal short repeat (IRS) region of the genome, e.g., an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome, of IRS-(i)-(ii)-(iii).
[0135] In some embodiments, the expression cassette further comprises polynucleotide(s) encoding a US 10 protein and/or a US 11 protein, e.g., as described above. In some such embodiments, the expression cassete comprises (i) the polynucleotide encoding IL- 12, (ii) the polynucleotide encoding the CD40 agonist, (iii) the polynucleotide encoding the CTLA-4 binding protein, and (iv) the polynucleotides encoding the US 10 protein and/or US 11 protein. In some embodiments, the expression cassete is in the orientation relative to the internal short repeat (IRS) region of the genome, e.g., an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome, of IRS-(i)-(ii)-(iii)-(iv). In other embodiments, the expression cassette further comprises a polynucleotide encoding a US 10 protein and a US 11 protein, e.g., as described above. In some such embodiments, the expression cassette comprises (i) the polynucleotide encoding IL- 12, (ii) the polynucleotide encoding the CD40 agonist, (iii) the polynucleotide encoding the CTLA-4 binding protein, and (iv) the polynucleotide encoding the US 10 and US 11 proteins. In some embodiments, the expression cassete is in the orientation relative to the internal short repeat (IRS) region of the genome, e.g., an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome, of IRS-(i)-(ii)-(iii)-(iv). In other embodiments, the expression cassette further comprises a polynucleotide encoding a US10 protein and a polynucleotide encoding a a US11 protein, e.g., as described above. In some such embodiments, the expression cassete comprises (i) the polynucleotide encoding IL- 12, (ii) the polynucleotide encoding the CD40 agonist, (iii) the polynucleotide encoding the CTLA-4 binding protein, (iv) the polynucleotide encoding the US 10 protein, and (v) the polynucleotide encoding the US 11 protein. In some embodiments, the expression cassete is in the orientation relative to the internal short repeat (IRS) region of the genome, e.g., an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome, ofIRS-(i)-(ii)-(iii)- (iv)-(v).
[0136] In some embodiments, the expression cassete further comprises a polynucleotide comprising a variant US11 gene, e.g., as described above. In some such embodiments, the expression cassette comprises: (i) the polynucleotide encoding IL-12, (ii) the polynucleotide encoding the CD40 agonist, (iii) the polynucleotide encoding the CTLA-4 binding protein, (iv) the polynucleotide(s) encoding the US 10 protein and/or US11 protein, or the polynucleotide encoding the US 10 and US 11 proteins, and (v) the polynucleotide comprising the variant US 11 gene. In some embodiments, the expression cassette is in the orientation relative to the internal short repeat (IRS) region of the genome, e.g., an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome, of IRS-(i)-(ii)-(iii)-(iv)-(v). In other embodiments, the expression cassette comprises a polynucleotide comprising a variant US 11 gene, e.g., as described above. In some such embodiments, the expression cassette comprises: (i) the polynucleotide encoding IL-12, (ii) the polynucleotide encoding the CD40 agonist, (iii) the polynucleotide encoding the CTLA-4 binding protein, (iv) the polynucleotide encoding the US 10 protein, (v) the polynucleotide encoding the US 11 protein, (vi) the polynucleotide comprising the variant US 11 gene. In some embodiments, the expression cassette is in the orientation relative to the internal short repeat (IRS) region of the genome, e.g., an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome, of IRS-(i)-(ii)-(iii)- (iv)-(v)-(vi).
V-B. Expression cassettes encoding FLT3L and/or TAP inhibitor
[0137] Also provided herein are expression cassettes comprising a polynucleotide encoding FLT3L and/or a polynucleotide encoding a transporter associated with antigen processing (TAP) inhibitor.
[0138] In some embodiments, an expression cassette of the disclosure comprises a promoter operably linked to the polynucleotide encoding FLT3L and/or to the polynucleotide encoding the TAP inhibitor. Any suitable promoter may be used in the cassettes of the disclosure, so long as the promoter drives expression of the associated polynucleotide. Exemplary and non-limiting promoters that may be used include the human cytomegalovirus (hCMV) promoter, the murine cytomegalovirus (mCMV) promoter, the Aotine betaherpesvirus 1 (AoHV 1) promoter, the CAG promoter, a CMV hybrid promoter, the EF1a promoter, the MMLV 5’ long terminal repeat (LTR) from the Moloney murine leukemia virus promoter (i.e., the MMLV promoter), the Pbidir3 promoter, and a native HSV promoter sequence.
[0139] In some embodiments, the expression cassette further comprises a polyadenylation signal operably linked to the polynucleotide encoding FLT3L and/or the polynucleotide encoding the TAP inhibitor. Any suitable polyadenylation signal may be used in the cassettes of the disclosure. Exemplary and non-limiting polyadenylation signals (polyA or pA) that may be used include the simian vacuolating virus 40 polyA (SV40pA), the human beta globin polyA (hBGpA), the human growth hormone polyA (hGH polyA), the rabbit beta globin polyA (rBGpA), a bovine growth hormone polyadenylation (BGHpA), a polyA derived from the human GAPDH gene, and a native HSV polyA sequence.
[0140] In some embodiments, the expression cassettes of the disclosure may comprise any suitable promoter and/or polyadenylation signal known in the art or described herein operably linked to any of the polynucleotide encoding FLT3L and/or to the polynucleotide encoding the TAP inhibitor.
[0141] In some embodiments, the TAP inhibitor is derived from herpes virus 1 or herpes virus 2. In some embodiments, the TAP inhibitor is derived from bovine herpes virus 1. In some embodiments, the TAP inhibitor is any of UL49.5, US6, or ICP47. In some embodiments, the TAP inhibitor is UL49.5
[0142] In some embodiments, the expression cassette further comprises a polynucleotide encoding a self-cleaving peptide. Any suitable self-cleaving peptide may be used in the cassettes of the disclosure, including, but not limited to, a T2A, P2A, E2A, or F2A peptide. In some embodiments, the encoded self-cleaving peptide is a P2A peptide. In some embodiments, the encoded P2A comprises the amino acid sequence of SEQ ID NO:91In some embodiments, the self-cleaving peptide is positioned between the polynucleotide encoding the FLT3L and the polynucleotide encoding the TAP inhibitor in the expression cassette.
[0143] In some embodiments, the expression cassette comprises a promoter operably linked to the polynucleotide encoding the FLT3L. In some embodiments, the promoter is the hCMV promoter. In some embodiments, the hCMV promoter comprises the nucleotide sequence of SEQ ID NO: 107 [0144] In some embodiments, the expression cassette further comprises a polyadenylation signal operably linked to the polynucleotide encoding the TAP inhibitor. In some embodiments, the polyadenylation sequence is a BGHpA polyadenylation signal. In some embodiments, the BGHpA polyadenylation signal comprises the nucleotide sequence of SEQ ID NO: 102.
[0145] In some embodiments, an expression cassette of the disclosure comprises, in order, a promoter (e.g., an hCMV promoter) operably linked to the polynucleotide encoding the FLT3L; the polynucleotide encoding the FLT3L; the polynucleotide encoding the self-cleaving peptide (e.g., a P2A peptide); the polynucleotide encoding the TAP inhibitor (e.g., a UL49.5 protein); and a polyadenylation signal (e.g., a BGHpA polyadenylation signal).
[0146] In some embodiments, an expression cassette of the disclosure comprises a polynucleotide encoding, in order, the FLT3L, the self-cleaving peptide (e.g., a P2A peptide), and the TAP inhibitor (e.g., a UL49.5 protein). In some embodiments, said polynucleotide comprises the nucleotide sequence of SEQ ID NO: 106. In some embodiments, the expression cassette encodes a polypeptide comprising, in order, the FLT3L, the self-cleaving peptide (e.g., a P2A peptide), and the TAP inhibitor (e.g., a UL49.5 protein). In some embodiments, the expression cassette further comprises a promoter, e.g., an hCMV promoter, that regulates expression of the polynucleotide encoding the FLT3L, the self-cleaving peptide (e.g., a P2A peptide), and the TAP inhibitor (e.g., a UL49.5 protein). In some embodiments, the expression cassette further comprises a polyadenylation signal, e.g., a BGHpA. In some embodiments, the expression cassette comprises, in order, a promoter, e.g., an hCMV promoter; a polynucleotide encoding, in order, the FLT3L, the self-cleaving peptide (e.g., a P2A peptide), and the TAP inhibitor (e.g., a UL49.5 protein); and a polyadenylation signal, e.g., a BGHpA.
[0147] In some embodiments, an expression cassette of the disclosure comprises the nucleotide sequence of SEQ ID NO: 100, or a nucleotide sequence having any of about at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the nucleotide sequence set forth in SEQ ID NO: 100.
[0148] In some embodiments, an expression cassette of the disclosure is integrated into a genome of a virus, such as an oncolytic HSV, e.g., an HSV-1 or HSV-2. In some embodiments, the cassette is integrated into one or two of the native γ34.5 loci of an oncolytic HSV, e.g., an HSV-1 or HSV- 2. In some embodiments, one or two of the native γ34.5 loci of an oncolytic HSV, e.g., an HSV-1 or HSV-2, are rendered inactive by insertion of the expression cassette. In some embodiments, integration of the expression cassette into a γ34.5 locus comprises replacing all or a part of the native γ34.5 locus with the expression cassette. In some embodiments, the TAP inhibitor encoded by the expression cassette is expressed as an immediate-early gene during viral replication.
[0149] In some embodiments, the expression cassette comprises: (i) the polynucleotide encoding the TAP inhibitor (e.g., a UL49.5 protein), (ii) the polynucleotide encoding the self-cleaving peptide (such as a P2A peptide), and (iii) the polynucleotide encoding the FLT3L, e.g., as described above. In some embodiments, the expression cassette is integrated in (e.g., replaces) the native γ34.5 locus within the long terminal repeat (TRL) region of the genome, e.g. an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome. In some embodiments, the expression cassette is in the orientation relative to the unique long (UL) region of the genome, e.g. an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome, of (i)-(ii)-(iii)-UL.
[0150] In some embodiments, the expression cassette comprises: (i) the polynucleotide encoding the TAP inhibitor (e.g., a UL49.5 protein), (ii) the polynucleotide encoding the self-cleaving peptide (e.g., a P2A peptide), and (iii) the polynucleotide encoding the FLT3L, e.g., as described above. In some embodiments, the expression cassette is integrated in (e.g., replaces) the native γ34.5 locus within the internal long repeat (IRL) region of the genome, e.g. an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome. In some embodiments, the expression cassette is in the orientation relative to the unique long (UL) region of the genome, e.g. an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome, of UL-(iii)-(ii)-(i).
V-C. Oncolytic viruses, genomes, vectors and cells comprising one or more expression cassettes
[0151] Also provided herein is an oncolytic virus (e.g., an oncolytic HSV, such as an oncolytic HSV-1 or oncolytic HSV-2) comprising one or more of the expression cassettes described above (e.g., in Sections IV-A and/or IV-B). In some embodiments, the oncolytic virus comprises one or more expression cassettes comprising a polynucleotide encoding IL- 12, a polynucleotide encoding a CD40 agonist, and/or a polynucleotide encoding a CTLA-4 binding protein, e.g., as described above in Section IV-A. In some embodiments, the oncolytic virus comprises one or more expression cassettes comprising a polynucleotide encoding FLT3L and/or a polynucleotide encoding a transporter associated with antigen processing (TAP) inhibitor, e.g., as described above in Section IV-B. In some embodiments, the oncolytic virus comprises: (a) one or more expression cassettes comprising a polynucleotide encoding IL- 12, a polynucleotide encoding a CD40 agonist, and/or a polynucleotide encoding a CTLA-4 binding protein, e.g., as described above in Section IV-A; and (b) one or more expression cassettes comprising a polynucleotide encoding FLT3L and/or a polynucleotide encoding a transporter associated with antigen processing (TAP) inhibitor, e.g., as described above in Section IV-B. In some embodiments, an oncolytic virus of the disclosure exhibits increased T cell activation relative to an oncolytic virus lacking any one, any two, or all of the polynucleotides encoding the IL- 12 protein, the CD40 agonist, and the CTLA-4 binding protein. T cell activation may be assessed using any suitable method known in the art, such as using an in vitro IL-2 secretion assay. In some embodiments, an oncolytic virus of the disclosure has increased abscopal effect relative to an oncolytic virus lacking any one, any two, or any three of the FLT3L, the IL- 12, the CD40 agonist, and the CTLA-4 binding protein. Abscopal effect may be assessed using any suitable method known in the art, such as using an in vivo tumor or cancer animal model, herein. In some embodiments, an oncolytic virus of the disclosure is capable of evading an individual’s immune system. In some embodiments, an oncolytic virus of the disclosure reduces or impairs viral antigen loading onto histocompatibility complex (MHC) Class I molecules for display at the cell surface, thereby reducing adaptive immune responses to the virus.
[0152] Also provided herein, is a modified HSV genome (e.g., an HSV-1 or HSV-2 genome) comprising one or more of the expression cassettes described above (e.g., in Sections IV-A and IV-B). In some embodiments, the modified HSV genome comprises one or more expression cassettes comprising a polynucleotide encoding IL- 12, a polynucleotide encoding a CD40 agonist, and/or a polynucleotide encoding a CTLA-4 binding protein, e.g., as described above in Section IV-A. In some embodiments, the modified HSV genome comprises one or more expression cassettes comprising a polynucleotide encoding FLT3L and/or a polynucleotide encoding a transporter associated with antigen processing (TAP) inhibitor, e.g., as described above in Section IV-B. In some embodiments, the modified HSV genome comprises: (a) one or more expression cassettes comprising a polynucleotide encoding IL- 12, a polynucleotide encoding a CD40 agonist, and/or a polynucleotide encoding a CTLA-4 binding protein, e.g., as described above in Section IV-A; and (b) one or more expression cassettes comprising a polynucleotide encoding FLT3L and/or a polynucleotide encoding a transporter associated with antigen processing (TAP) inhibitor, e.g., as described above in Section IV-B.
[0153] Also provided herein, is a vector comprising one or more of the expression cassettes described above (e.g., in Sections IV-A and IV-B). Suitable vectors include, without limitation, cloning vectors and expression vectors. Suitable cloning vectors can be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors generally have the ability to self -replicate, may possess a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in selecting clones containing the vector. Suitable examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR.322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors such as BioRad, Strategene, and Invitrogen. Expression vectors generally are replicable polynucleotide constructs that contain a nucleic acid of the present disclosure. The expression vector may be replicable in the host cells either as episomes or as an integral part of the chromosomal DNA. Suitable expression vectors include but are not limited to plasmids, viral vectors, including adenoviruses, adeno-associated viruses, HSV viruses, e.g. HSV-1 or HSV-2, retroviruses, and cosmids. Vector components may generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcriptional controlling elements (such as promoters, enhancers and terminator). For expression (i.e., translation), one or more translational controlling elements are also usually included, such as ribosome binding sites, translation initiation sites, and stop codons.
[0154] In some embodiments, cells, such as host cells, comprising one or more of the expression cassettes described above (e.g., in Sections IV-A and IV-B) are also provided. In some embodiments, the cell is an isolated cell. An isolated cell is a cell that is identified and separated from at least one contaminant cell with which it is ordinarily associated in the environment in which it was produced. In some embodiments, the isolated cell is free of association with all components associated with the production environment. The isolated cell is in a form other than in the form or setting in which it is found in nature. Isolated cells are distinguished from cells existing naturally in tissues, organs, or individuals. In some embodiments, the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell, human cells such as HELA cells, HEK293 cells, etc., or lymphoid cell (e.g., Y0, NSO, Sp20 cell). Host cells of the present disclosure also include, without limitation, isolated cells, in vitro cultured cells, and ex vivo cultured cells. In some embodiments, the cell is a mammalian cell.
VI. JP-OV-2
[0155] JP-OV-2 is a next-generation recombinant HSV Type-1-based oncolytic virus (OV) that has been modified to 1) reduce innate and adaptive antiviral host responses that shut down viral replication to allow greater lysis of target tumor cells and 2) to enhance all stages of the adaptive immune response to the cancer cells through expression of multiple synergistic immune stimulatory payloads.
[0156] In some embodiments, JP-OV-2 comprises: a. a cassette integrated in one or both of the γ34.5 loci comprising in order, from upstream to downstream, a CMV promoter, a polynucleotide encoding hFLT3L, a P2A cleavage sequence, a polynucleotide encoding UL49.5, and a polyadenylation signal; and b. another cassette integrated in the US 10- 12 locus comprising in order, from upstream to downstream, a polynucleotide comprising a variant US11 gene encoding native US11 protein, an additional polynucleotide encoding native US11 protein, a polynucleotide encoding US 10 protein, a polyadenylation signal that is operably linked to the polynucleotide encoding the US 10 protein, a CMV promoter, a polynucleotide encoding a CTLA-4 binding protein, a polyadenylation signal that is operably linked to the polynucleotide encoding the CTLA-4 binding protein, a polyadenylation signal that is operably linked to a polynucleotide encoding a CD40 agonist, a polynucleotide encoding a CD40 agonist, an AoHVl promoter that controls expression of the CD40 agonist, an MMLV promoter that controls expression of an IL- 12, a polynucleotide encoding an IL- 12, and a polyadenylation signal that is operably linked to the polynucleotide encoding the IL- 12.
[0157] In some embodiments, JP-OV-2 comprises a polynucleotide for hFLT3L encoding the amino acid sequence set forth in SEQ ID NO:71.
[0158] In some embodiments, JP-OV-2 comprises a polynucleotide for UL49.5 encoding the amino acid sequence set forth in SEQ ID NO: 82.
[0159] In some embodiments, JP-OV-2 comprises a polynucleotide for IL- 12 encoding the amino acid sequence set forth in SEQ ID NO: 4.
[0160] In some embodiments, JP-OV-2 comprises a polynucleotide for CD40 agonist encoding the amino acid sequence set forth in SEQ ID NO: 25,
[0161] In some embodiments, JP-OV-2 comprises a polynucleotide for CTLA-4 binding protein encoding the amino acid sequence set forth in SEQ ID NO: 50.
[0162] In some embodiments, JP-OV-2 comprises a polynucleotide for variant US11 gene comprising the polynucleotide sequence set forth in SEQ ID NO: 204.
[0163] In some embodiments, JP-OV-2 comprises an additional polynucleotide encoding for US11 encoding the amino acid sequence set forth in SEQ ID NO: 80.
[0164] In some embodiments, JP-OV-2 comprises a polynucleotide for US 10 encoding the amino acid sequence set forth in SEQ ID NO:90. VII. Methods of making oncolytic viruses
[0165] The oncolytic viruses (such as the oncolytic HSV) described herein may be prepared using any methods known in the art or as described herein. In some embodiments, the oncolytic virus (such as the oncolytic HSV) may be engineered (such as to comprise one or more of the expression cassettes described herein and/or to express one or more of the pay load proteins described herein) by modifying a wild-type virus (such as a wild-type HSV-1) genome. Transgenes and/or expression cassettes, including as otherwise described herein, may be inserted in the native genome or replace native portions of the genome using recombinant cloning techniques well known in the art. Exemplary engineering methods are described herein at Examples 4-7. Engineered oncolytic virus genomes may be propagated in suitable cells and collected from cell media or collected from cell lysates. The virus-containing cell media or virus-containing cell lysates may then be sterilized, such as by filtration or other suitable means. The virus may be concentrated, such as by ultracentrifugation. Purified virus may be stored by suitable means, including by storage at about -80 °C in DMEM. Titers of virus stocks vary by orders of magnitude, depending upon the viral genotype and the protocol used to prepare and purify them. Purified virus may be titered using assays well known in the art. Viral titer may be expressed in terms of infectious viral units, such as plaque-forming units (pfu). The integrity and sequence of the viral genome may be assessed by techniques well known in the art, including whole-genome sequencing.
VIII. Methods of treatment
[0166] The various aspects and embodiments described in this section in the context of a method of treatment also apply to an oncolytic herpes simplex type 1 (HSV-1) virus for use according to the methods described herein unless indicated otherwise. Similarly, the various aspects and embodiments described in this section in the context of a method of treatment also apply to an oncolytic herpes simplex type 1 (HSV-1) virus in combination with a PD-1 antibody, according to the methods described herein.
A. Patient Populations
[0167] The present disclosure in some aspects provides a method of treating an advanced solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual every two weeks (Q2W) intratumorally. In some embodiments, the HSV-1 virus is JP-OV-2. In some embodiments, the individual has an advanced tumor. In some embodiments, the individual has a metastatic solid tumor. In some embodiments, the individual does not have a central nervous system solid tumor.
[0168] In some embodiments, the individual has previously received available standard therapy and has progressed. In some embodiments, progressive disease is defined as an increase in the sum of lesion diameters ≥20% and ≥5 mm from nadir. In some embodiments, the individual cannot tolerate standard therapy. In some embodiments, the individual has refused standard therapy. In some embodiments, the individual has an advanced or metastatic solid tumor for which there is no standard of care per regional guidelines.
[0169] In some embodiments, the individual has at least 1 injectable tumor ≥1 cm in longest diameter (or shortest diameter for lymph nodes). In some embodiments, the individual has injectable tumors that in aggregate are ≥1 cm in the longest diameter. In some embodiments, the injectable tumors is measurable by RECIST v1.1 criteria. In some embodiments, the injectable lesion is not invading or in close proximity to major or large blood vessels. In some embodiments, the injectable lesion is not invading major airways.
[0170] In some embodiments, the individual has at least one measurable lesion that will not be injected during the treatment.
[0171] In some embodiments, the individual has a histologically or cytologically confirmed non- small cell lung cancer (NSCLC). In some embodiments, the individual has stage IIIB-IV NSCLC according to the 8th edition of the American Joint Committee on Cancer (AJCC) TNM staging for NSCLC. In some embodiments, the TNM staging system for NSCLC comprises T for characteristics of the primary tumor, N for nodal involvement, and M for (distant) metastasis. In some embodiments, specific T, N, and M categories that exhibit similar behavior are coalesced into stage groups.
[0172] In some embodiments, the individual has stage IIIB NSCLC. In some embodiments, stage IIIB NSCLC is selected from the group consisting of T1b/N3/M0, T1c/N3/M0, T2a/N3/M0, T2b/N3/M0, T3/N2/M0, and T4/N2/M0 NSCLC. In some embodiments, the individual has stage IIIC NSCLC. In some embodiments, stage IIIC NSCLC comprises T4/N3/M0 NSCLC. In some embodiments, the individual has stage IVA NSCLC. In some embodiments the stage IVA NSCLC comprises Tany/Nany/M1a NSCLC, or Tany/Nany/M1b NSCLC. In some embodiments, the individual has stage IVB NSCLC. In some embodiments, the stage IVB NSCLC comprises Tany/Nany/Mlc NSCLC.
[0173] In some embodiments, the individual with histologically or cytologically confirmed NSCLC, stage IIIB-IV has been previously treated with an anti-PD-1/PD-L1 therapy and platinum- based chemotherapy, either as combination or sequentially for metastatic disease and has progressed on or after therapy. In some embodiments, the individual cannot tolerate or has previously refused platinum-based chemotherapy or were unable to receive platinum-based chemotherapy have progressed after anti-PD-1/PD-L1 therapy alone. In some embodiments, the stage IIIB-IV NSCLC is relapsed NSCLC. In some embodiments, the stage IIIB-IV NSCLC is refractory NSCLC.
[0174] In some embodiments, the individual has an ECOG performance status of Grade 0 or 1. In some embodiments, the individual has an ECOG performance status of Grade 0. In some embodiments, the individual with an ECOG performance status of Grade 0 is fully active and able to carry on all pre-disease performance without restriction. In some embodiments, the individual has an ECOG performance status of Grade 1. In some embodiments, the individual with an ECOG performance status of Grade 1 is restricted in physically strenuous activity but ambulatory and able to carry out work of a light or sedentary nature.
[0175] In some embodiments, the individual does not have active disease involvement of the central nervous system. In some embodiments, the active disease involvement of the central nervous system comprises primary central nervous system tumors, metastases, or leptomeningeal disease. In some embodiments, the individual has brain metastases that are definitively, locally treated, clinically stable and asymptomatic for more than two weeks, and who are not receiving steroids or receiving low dose corticosteroid treatment for at least 2 weeks prior to treatment with the oncolytic herpes simplex type 1 (HSV-1) virus. In some embodiments, low does corticosteroid treatment comprises less than about 10 mg prednisone or equivalent.
[0176] In some embodiments, the individual does not have prior or concurrent second malignancy that due to natural history or treatment is likely to interfere with treatment with the oncolytic herpes simplex type 1 (HSV-1) virus.
[0177] In some embodiments, the individual does not have an active herpetic infection. In some embodiments, the individual does not have a prior history of herpetic infection. In some embodiments, the individual does not have active herpetic infections that require ongoing systemic anti-viral therapy. In some embodiments, herpetic infections comprise herpetic keratitis or encephalitis.
[0178] In some embodiments, the individual does not have an active infection or condition that requires treatment with systemic anti-infective agents within about 7 days prior to the first dose of an oncolytic herpes simplex type 1 (HSV-1) virus. In some embodiments, the individual does not have an active infection or condition that requires chronic treatment with systemic anti-infective agents. In some embodiments, anti-infective agents comprise antibiotics, antifungals, or antivirals. [0179] In some embodiments, the individual does not have active autoimmune disease that requires systemic immunosuppressive medications within 12 months prior to treatment with an oncolytic herpes simplex type 1 (HSV-1) virus. In some embodiments, systemic immunosuppressive medications comprise chronic corticosteroid, methotrexate, or tacrolimus.
[0180] In some embodiments, the individual is not immunocompromised. In some embodiments, the individual does not have a known positive test result for HIV or other immunodeficiency syndrome.
[0181] In some embodiments, the individual does not have a history of non- infectious pneumonitis that required systemic treatment with corticosteroids. In some embodiments, the individual does not have a history of interstitial lung disease that required systemic treatment with corticosteroids. In some embodiments, the individual does not have a history of non-infectious pneumonitis that required continuous supplemental oxygen use to maintain adequate oxygenation. In some embodiments, the individual does not have a history of interstitial lung disease that required continuous supplemental oxygen use to maintain adequate oxygenation.
[0182] In some embodiments, the individual does not have a history of solid organ transplantation. In some embodiments, the individual does not have a history of hematologic stem cell transplantation.
[0183] In some embodiments, the individual does not have an active bleeding diathesis or requirement for therapeutic anticoagulation that cannot be interrupted or altered for procedures.
[0184] In some embodiments the individual does not have venous thromboembolic events within 1 month prior to the first dose of an oncolytic herpes simplex type 1 (HSV-1) virus. In some embodiments, the venous thromboembolic event comprises a pulmonary embolism.
[0185] In some embodiments, the individual does not have clinically significant cardiovascular disease within 6 months prior to treatment with the oncolytic herpes simplex type 1 (HSV-1) virus. In some embodiments, the clinically significant cardiovascular disease is selected from the group consisting of myocardial infarction, severe or unstable angina, or coronary artery bypass surgery, clinically significant arrhythmias, congestive heart failure such as NYHA class III or V, cerebrovascular accident, transient ischemic attack, or other arterial thromboembolic event, and Myocarditis.
[0186] In some embodiments, the individual has not had prior treatment with an HSV-based oncolytic virus for the treatment of metastatic disease.
[0187] In some embodiments, the individual has not received prior anti-PD(L)-1 or anti-CTLA-4 therapy within 4 weeks, or other anti cancer therapy within 14 days, before the first dose of HSV treatment.
[0188] In some embodiments, the individual has not received radiation therapy within 7 days before the first dose of HSV treatment.
[0189] In some embodiments, the individual has not received immunosuppressive doses of systemic medication, such as corticosteroids within 7 days prior to the first dose of HSV treatment. [0190] In some embodiments, the individual has not received any live vaccine within 28 days before the first dose of HSV treatment.
B. Dosing Regimens
[0191] The following section describes various aspects (embodiments) of dosing and treatment regimens, any and all of which apply to the methods described herein. The various aspects and embodiments described in this section in the context of a method of treatment also apply to an oncolytic herpes simplex type 1 (HSV-1) virus for use according to the methods described herein unless indicated otherwise.
[0192] The present application in one aspect provides a method of treating an advanced solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual every two weeks (Q2W) intratumorally. In some embodiments, the oncolytic virus comprises one or more expression cassettes comprising a polynucleotide encoding hFLT3L protein, a polynucleotide encoding a CTLA-4 binding protein, a polynucleotide encoding a CD40 agonist, and a polynucleotide encoding IL- 12. In some embodiments the HSV-1 virus is JP-OV- 2. In some embodiments, the method further comprises administering an antibody that binds to PD-1 every four weeks (Q4W). In some embodiments, the antibody that binds to PD-1 is administered intravenously. In some embodiments, the antibody that binds to PD-1 is administered to the individual at a dose of about 480 mg. In some embodiments, the antibody that binds to PD-
1 is cetrelimab. In some embodiments, about 480 mg of cetrelimab is administered Q4W intravenously to the individual.
[0193] In some embodiments, the present application in one aspect provides a method of treating an advanced solid tumor in an individual comprising administering an oncolytic HSV-1 virus to the individual intratumorally Q2W and administering an antibody that binds to PD-1 to the individual intravenously Q4W, wherein the first administration of the HSV-1 virus occurs on week
1 and the first administration of the PD-1 antibody occurs on week 3. In some embodiments, the
HSV-1 virus is JP-OV-2. In some embodiments, the HSV-1 virus is administered at a dose of about
105 PFU/mL, about 106 PFU/mL, about 107 PFU/mL, or about 108 PFU/mL. In some embodiments, the HSV-1 virus is administered at a dose of about 105 PFU/mL. In some embodiments, the HSV-1 virus is administered at a dose of about 106 PFU/mL. In some embodiments, the HSV-1 virus is administered at a dose of about 107 PFU/mL. In some embodiments, the HSV-1 virus is administered at a dose of about 108 PFU/mL. In some embodiments, the antibody that binds to PD-1 is cetrelimab. In some embodiments, about 480 mg of cetrelimab is administered intravenously to the individual during each administration. In some embodiments, about 480 mg of cetrelimab is administered intravenously to the individual during each administration and after administration of the HSV-1 virus.
[0194] In some embodiments, provided herein is a method of treating an advanced or metastatic non-central nervous system tumor in an individual comprising administering about 105 PFU/mL, about 106 PFU/mL, about 107 PFU/mL, or about 108 PFU/mL of an HSV-1 virus and administering an antibody that binds to PD-1 to the individual. In some embodiments, the HSV-1 virus is administered at a dose of about 105 PFU/mL. In some embodiments, the HSV-1 virus is administered at a dose of about 106 PFU/mL. In some embodiments, the HSV-1 virus is administered at a dose of about 107 PFU/mL. In some embodiments, the HSV-1 virus is administered at a dose of about 108 PFU/mL. In some embodiments, the individual has received all available standard therapies and progressed. In some embodiments, the HSV-1 virus is JP-OV- 2. In some embodiments, the HSV-1 virus is administered via intratumoral injection. In some embodiments, the individual is administered the HSV-1 virus via a superficial approach comprising injection under direct visualization and palpation into a cutaneous, subcutaneous, or nodal lesion. In some embodiments, the individual is administered the HSV-1 virus via a percutaneous approach for lesions accessible via imaging guidance. In some embodiments, the percutaneous administration approach is used for transthoracically accessible lung lesions, transabdominally accessible liver lesions, or subcutaneous soft tissue lesions.
[0195] In some embodiments, the HSV-1 virus is administered to the individual intratumorally Q2W between about 1 to about 8 times.. In some embodiments, the HSV-1 virus is administered to the individual 1, 2, 3, 4, 5, 6, 7, or 8 times. In some embodiments, the HSV-1 virus is JP-OV- 2.
[0196] In some embodiments, the individual receives intravenous administration of an antibody that binds to PD-1 Q4W for up to a total of about two years. In some embodiments the antibody that binds to PD-1 is cetrelimab.
[0197] In some embodiments, provided herein is a method of treating an advanced solid tumor in an individual comprising administering an oncolytic HSV-1 virus to the individual intratumorally Q2W and administering an antibody that binds to PD-1 to the individual intravenously Q4W, wherein the first administration of the HSV-1 virus occurs on week 1 and the first administration of the PD-1 antibody occurs on week 3. In some embodiments, the HSV-1 virus is JP-OV-2. In some embodiments the antibody that binds to PD-1 is cetrelimab. In some embodiments, the individual has an advanced or metastatic non-central nervous system tumor. In some embodiments, the individual has received all available standard therapies and progressed. In some embodiments, the method of treating a solid tumor in an individual comprises i) administering an HSV-1 virus intratumorally Q2W on weeks 1, 3, 5, 7, 9, 11, 13, and 15, and ii) administering an antibody that binds to PD-1 intravenously Q4W on weeks 3, 7, 11, and 15.
[0198] In some embodiments, the present application in one aspect provides a method of treating advanced metastatic NSCLC in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual intratumorally Q2W and administering an antibody that binds to PD-1 to the individual intravenously Q4W, wherein the first administration of the HSV-1 virus and the first administration of the PD-1 antibody occurs on week 1. In some embodiments, the HSV-1 virus is administered at a dose of about 105 PFU/mL, about 106 PFU/mL, about 107 PFU/mL, or about 108 PFU/mL. In some embodiments, the HSV-1 virus is administered at a dose of about 105 PFU/mL. In some embodiments, the HSV-1 virus is administered at a dose of about 106 PFU/mL. In some embodiments, the HSV-1 virus is administered at a dose of about 107 PFU/m. In some embodiments, the HSV-1 virus is administered at a dose of about 108 PFU/mL. In some embodiments the HSV-1 virus is JP-OV-2. In some embodiments, the antibody that binds to PD-1 is cetrelimab. In some embodiments, about 480 mg of cetrelimab is administered intravenously to the individual during each administration. In some embodiments, about 480 mg of cetrelimab is administered intravenously to the individual during each administration and after administration of the HSV-1 virus.
[0199] In some embodiments, provided herein is a method of treating advanced metastatic NSCLC in an individual comprising administering about 105 PFU/mL, about 106 PFU/mL, about 107 PFU/mL, or about 108 PFU/mL of an HSV-1 virus and administering an antibody that binds to PD-1 to the individual. In some embodiments, the HSV-1 virus is administered at a dose of about 105 PFU/mL. In some embodiments, the HSV-1 virus is administered at a dose of about 106 PFU/mL. In some embodiments, the HSV-1 virus is administered at a dose of about 107 PFU/m. In some embodiments, the HSV-1 virus is administered at a dose of about 108 PFU/mL. In some embodiments the HSV-1 virus is JP-OV-2. In some embodiments, the individual has progressed after treatment with an anti-PD-1 antibody and chemotherapy. In some embodiments, the individual has stage IIIB-IV NSCLC according to the 8th edition of the American Joint Committee on Cancer (AJCC) TNM staging for NSCLC. In some embodiments, the individual has at least 2 measurable lesions, a first measurable lesion that is accessible for endobronchial or percutaneous injection, and a second measurable lesion that will not be injected. In some embodiments, the individual has a maximum of 5 measurable lesions in total at the start of treatment. In some embodiments, the individual does not have more than 2 measurable lesions per organ at the start of treatment. In some embodiments, the HSV-1 virus is administered intratumorally Q2W. In some embodiments the PD-1 antibody is administered intravenously Q4W.
[0200] In some embodiments, the HSV-1 virus is administered to an individual with NSCLC via intratumoral injection. In some embodiments, the individual is administered the HSV-1 virus via a superficial approach with injection under direct visualization and palpation into a cutaneous, subcutaneous, or nodal lesion. In some embodiments, the individual is administered the HSV-1 virus via a percutaneous approach for lesions accessible via imaging guidance. In some embodiments, the percutaneous administration approach is selected for transthoracically accessible lung lesions, transabdominally accessible liver lesions, or subcutaneous soft tissue lesions. In some embodiments, the individual is administered the HSV-1 virus via an endobronchial approach for lung lesions and lymph nodes accessible via a bronchoscope. In some embodiments the HSV-1 virus is JP-OV-2.
[0201] In some embodiments, the HSV-1 virus is administered to an individual with NSCLC intratumorally Q2W between about 1 to about 8 times. In some embodiments, the HSV-1 virus is administered to the individual 1, 2, 3, 4, 5, 6, 7, or 8 times. In some embodiments, the HSV-1 virus is administered to the individual 8 times. In some embodiments, the individual receives at most 8 treatments with the HSV-1 virus. In some embodiments, the individual is administered the HSV- 1 virus intratumorally Q2W for between about 2 and about 16 weeks. In some embodiments, the individual is administered the HSV-1 virus intratumorally Q2W for about 2 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 14 weeks or about 16 weeks. In some embodiments, the individual is administered the HSV-1 virus intratumorally Q2W for about 16 weeks. In some embodiments the HSV-1 virus is JP-OV-2.
[0202] In some embodiments, the individual receives intravenous administration of an antibody that binds to PD-1 Q4W for up to a total of about two years. In some embodiments, the antibody that binds to PD-1 is cetrelimab.
[0203] In some embodiments, the present application in one aspect provides a method of treating advanced metastatic NSCLC in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual intratumorally Q2W and administering an antibody that binds to PD-1 to the individual intravenously Q4W, wherein the first administration of the HSV-1 virus and the first administration of the PD-1 antibody occurs on week 1. In some embodiments the HSV-1 virus is JP-OV-2. In some embodiments, the antibody that binds to PF-1 is cetrelimab. In some embodiments, the individual has progressed after treatment with an anti- PD-1 antibody and chemotherapy. In some embodiments, the individual has stage IIIB-IV NSCLC according to the 8th edition of the American Joint Committee on Cancer (AJCC) TNM staging for NSCLC. In some embodiments, the method of treating advanced metastatic NSCLC in an individual comprises i) administering an HSV-1 virus intratumorally Q2W on weeks 1, 3, 5, 7, 9, 11, 13, and 15, and ii) administering an antibody that binds to PD-1 intravenously Q4W on weeks 1, 5, 9, 13, and 15.
[0204] In some embodiments, the HSV-1 virus is administered in a maximum volume of aboutlO ml. In some embodiments, the HSV-1 virus is administered in an injection volume calculated based on lesion size. In some embodiments, the HSV-1 virus is administered to a superficial lesion or a visceral lesion. In some embodiments, the HSV-1 virus is administered in a volume of about 4 ml for a superficial lesion comprising a lesion size greater than at least 5 cm in the longest dimension. In some embodiments, the HSV-1 virus is administered in a volume of about 3 ml for a superficial lesion comprising a lesion size in the longest dimension of between about 4 cm and about 5 cm. In some embodiments, the HSV-1 virus is administered in a volume of about 2 ml for a superficial lesion comprising a lesion size in the longest dimension of between about 3 cm and about 4 cm. In some embodiments, the HSV-1 virus is administered in a volume of about 1.5 ml for a superficial lesion comprising a lesion size in the longest dimension of between about 2 cm and about 3 cm. In some embodiments, the HSV-1 virus is administered in a volume of about 0.5 ml for a superficial lesion comprising a lesion size in the longest dimension of between about 1.5 cm and about 2 cm. In some embodiments, the HSV-1 virus is administered in a volume of about 0.3 ml for a superficial lesion comprising a lesion size in the longest dimension of between about 1 cm and about 1.5 cm. In some embodiments, the HSV-1 virus is administered in a volume of about 0.2 ml for a superficial lesion comprising a lesion size in the longest dimension of less than about 1 cm. In some embodiments, large superficial lesions may be injected using a corresponding visceral injection volume for the size of the given superficial lesion. In some embodiments, the full amount of HSV-1 virus cannot be administered to the lesion due to changes in lesion size, for example a lesion may decrease in size following a previous disease assessment. In some embodiments, the pressure required to administer the full amount of HSV-1 virus to the lesion is prohibitive in the opinion of the treating physician. In some embodiments the HSV-1 virus is JP- OV-2.
[0205] In some embodiments, the HSV-1 virus is administered in a maximum volume of about 10 ml. In some embodiments, the HSV-1 virus is administered in an injection volume calculated based on lesion size. In some embodiments, the HSV-1 virus is administered to a superficial lesion or a visceral lesion. In some embodiments, the HSV-1 virus is administered in a volume of about 10 ml for a visceral lesion comprising a lesion size greater than at least 5 cm in the longest dimension. In some embodiments, the HSV-1 virus is administered in a volume of about 8 ml for a visceral lesion comprising a lesion size in the longest dimension of between about 4 cm and about 5 cm. In some embodiments, the HSV-1 virus is administered in a volume of about 6 ml for a visceral lesion comprising a lesion size in the longest dimension of between about 3.5 cm and about 4 cm. In some embodiments, the HSV-1 virus is administered in a volume of about 4 ml for a visceral lesion comprising a lesion size in the longest dimension of between about 2.5 cm and about 3.5 cm. In some embodiments, the HSV-1 virus is administered in a volume of about 2 ml for a visceral lesion comprising a lesion size in the longest dimension of between about 2 cm and about 2.5 cm. In some embodiments, the HSV-1 virus is administered in a volume of about 1 ml for a visceral lesion comprising a lesion size in the longest dimension of between about 1.5 cm and about 2 cm. In some embodiments, the HSV-1 virus is administered in a volume of about 0.5 ml for a visceral lesion comprising a lesion size in the longest dimension of between about 1 cm and about 1.5 cm. In some embodiments, the full amount of HSV-1 virus cannot be administered to the lesion due to changes in lesion size, for example a lesion may decrease in size following a previous disease assessment. In some embodiments, the pressure required to administer the full amount of HSV-1 virus to the lesion is prohibitive in the opinion of the treating physician. In some embodiments the HSV-1 virus is JP-OV-2.
[0206] In some embodiments, one or more lesions are injected with HSV-1 virus during each administration. In some embodiments, regardless of the number of one or more lesions injected, the maximum total HSV-1 dose volume injected across all lesions is no more than about 10 mL. In some embodiments the HSV-1 virus is JP-OV-2.
[0207] In some embodiments, lesions are selected for injection in an individual with NSCLC. In some embodiments, only lesions in one hemithorax can be injected per administration. In some embodiments, injections of the HSV-1 virus into the lung may involve endobronchial injection via a bronchoscope. In some embodiments, injections of the HSV-1 virus into the lung may involve percutaneous image-guided injections for lesions that are not accessible by a bronchoscope. In some embodiments, at least 1 lesion is selected as an uninjectable target lesion unless all other lesions become uninjectable. In some embodiments, during the first administration of HSV-1 virus the largest injectable lesion is selected as a target lesion for injection of the HSV-1 virus. In some embodiments, the largest injectable lung lesion is prioritized for injection. In some embodiments, primary and affected measurable lymph nodes are selected for injection subsequent to the largest injectable lung lesion.
[0208] In some embodiments, the HSV-1 virus is injected into a superficial lesion. In some embodiments, a superficial lesion comprises a cutaneous, subcutaneous, or nodal lesion that can be accessed in the clinic under direct visualization/palpation. In some embodiments, the HSV-1 virus is injected into a cutaneous, subcutaneous, or nodal lesion. In some embodiments the HSV- 1 virus is JP-OV-2.
[0209] In some embodiments, the HSV-1 virus is injected into a visceral lesion or a deep nodal lesion. In some embodiments the HSV-1 virus is JP-OV-2. In some embodiments, injecting the HSV-1 virus into a visceral lesion requires imaging guidance, for example ultrasound, CBCT, or CT imaging. In some embodiments, the HSV-1 virus is not injected into visceral lesions with major airway or blood vessel invasion. In some embodiments, the HSV-1 virus is not injected into visceral lesions with a history of poor wound healing. In some embodiments, no more than 3 visceral lesions within a single solid organ should be injected at a single administration.
[0210] In some embodiments, the HSV-1 virus is injected into a transthoracically accessible lung lesion, a transabdominally accessible liver lesion, and/or a subcutaneous soft tissue lesion. In some embodiments, the HSV-1 virus is administered to a lung lesion or a lymph node accessible via a bronchoscope. In some embodiments the HSV-1 virus is JP-OV-2.
[0211] In other aspects, the present application provides a method of treating an advanced solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual every two weeks (Q2W) intratumorally in combination with administration of an antibody that binds to PD-1 every four weeks (Q4W). In some embodiments the HSV-1 virus is JP-OV-2. In some embodiments, the antibody that binds to PD-1 is administered intravenously. In some embodiments, the antibody that binds to PD-1 is administered to the individual at a dose of about 480 mg. In some embodiments, the antibody that binds to PD-1 is cetrelimab. In some embodiments, about 480 mg of cetrelimab is administered intravenously to the individual about every 4 weeks (Q4W) for at least about 15 weeks.
[0212] In some embodiments, the antibody that specifically binds to PD-1 comprises a heavy chain and a light chain, wherein the heavy chain of the antibody comprises a heavy chain variable region (VH) comprising a CDR-H1, CDR-H2, and CDR-H3 of a VH comprising the amino acid sequence set forth in SEQ ID NO:400, and the light chain of the antibody comprises a light chain variable region (VL) comprising a CDR-L1, CDR-L2, and CDR-L3 of a VL comprising the amino acid sequence set forth in SEQ ID NO:404.
[0213] In some embodiments, the antibody that specifically binds to PD-1 comprises a VH comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 401, a CDR- H2 comprising the amino acid sequence set forth in SEQ ID NO: 402, a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 403; and/or a VL comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 405, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 406, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 407.
[0214] In some embodiments, the antibody that binds to PD-1 is administered to the individual every four weeks (Q4W). In some embodiments, the antibody that binds to PD-1 is administered to the individual every four weeks (Q4W) and after administration of the HSV-1 virus.
[0215] In some embodiments, provided herein is a method of treating a solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual about every two weeks (Q2W) intratumorally and administering an antibody that binds to PD-1 about every four weeks (Q4W) intravenously, for at least about 15 weeks. In some embodiments the HSV-1 virus is JP-OV-2. In some embodiments, the antibody that binds to PD-1 is cetrelimab.
[0216] In some embodiments, provided herein is a method of treating a solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual about every two weeks (Q2W) intratumorally for at least about 15 weeks and administering an antibody that binds to PD-1 about every four weeks (Q4W) intravenously, for at most 2 years. In some embodiments the HSV-1 virus is JP-OV-2. In some embodiments, the antibody that binds to PD- 1 is cetrelimab.
[0217] Alternative dosing regimens:
[0218] In some embodiments, once the first three dose escalation levels have been evaluated using a Q2W schedule for JP-OV-2, alternative dosing regimens for JP-OV-2 may be implemented following safety and efficacy evaluation. In some embodiments, these alternative dosing regimens may involve dose intensification.
[0219] In some embodiments, treatment with JP-OV-2 is initiated and continued in combination with cetrelimab, such that participants receive combination treatment starting with the first dose. In some embodiments, up to 8 treatments of JP-OV-2 may be administered, with the possibility of administering an additional 8 doses, provided that defined safety criteria are met.
[0220] In some embodiments, an intensified dosing regimen comprises JP-OV-2 administration on Days 1 and 8 of each 28-day cycle of cetrelimab. [0221] In some embodiments, an intensified regimen comprises JP-OV-2 administration within the first 7 days of each 28-day cycle of cetrelimab.
[0222] In some embodiments, higher ratios of injected JP-OV-2 will be achieved by limiting the largest lesion diameter for injection to 4 cm or less in order to administer a full 10 mL target JP- OV-2 dose. In some embodiments, the alternative injection volumes per lesion size are specified in Table 19, and these volumes may be evaluated on a Q2W schedule or through alternative dosing regimens.
C. Endpoints
[0223] The various aspects and embodiments described in this section in the context of a method of treatment also apply to an oncolytic herpes simplex type 1 (HSV-1) virus for use according to the methods described herein unless indicated otherwise.
[0224] In some embodiments, provided herein is a method of treating an advanced solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual every two weeks (Q2W) intratumorally, wherein the individual experiences tumor size reduction. In some embodiments, the HSV-1 is JP-OV-2. In some embodiments, the method further comprises administering an antibody that binds to PD-1 about every four weeks (Q4W) intravenously. In some embodiments, the antibody that binds to PD-1 is cetrelimab. In some embodiments, the individual has more than one solid tumor. In some embodiments, the HSV-1 virus is administered to at least one of the solid tumors. In some embodiments, change in tumor size is defined as the difference in percentage change in sum of diameters of injected lesions and uninjected lesions, as measured based on RECIST vl.1. In some embodiments, such treatment results in a reduction in the Sum of Diameter (SOD) in an individual. In some embodiments, the Sum of diameter (SOD) is defined as the sum of diameter of all lesions with measurement(s).
[0225] In some embodiments, the individual experiences an abscopal effect. In some embodiments, the size of a solid tumor other than the at least one solid tumor where the HSV-1 is administered decreases in size following treatment.
[0226] In some embodiments, provided herein is a method of treating an advanced solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual every two weeks (Q2W) intratumorally and administering an antibody that binds to PD- 1 about every four weeks (Q4W) intravenously, wherein the individual experiences a complete response. In some embodiments, an individual receiving such treatment experiences a partial response. In some embodiments, an individual receiving such treatment experiences stable disease. In some embodiments, the HSV-1 is JP-OV-2. In some embodiments, the antibody that binds to PD-1 is cetrelimab.
[0227] In some embodiments, provided herein is a method of treating an advanced solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual every two weeks (Q2W) intratumorally and administering an antibody that binds to PD- 1 about every four weeks (Q4W) intravenously, wherein such treatment results in an objective response rate (ORR) in a population of patients who have received such treatment. In some embodiments, the HSV-1 is JP-OV-2. In some embodiments, the antibody that binds to PD-1 is cetrelimab. In some embodiments, the Objective Response Rate (ORR) is defined as the proportion of participants who have best response of Complete Response (CR) or Partial Response (PR) according to RECIST v1.1.
[0228] In some embodiments, provided herein is a method of treating an advanced solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual every two weeks (Q2W) intratumorally and administering an antibody that binds to PD- 1 about every four weeks (Q4W) intravenously, wherein such treatment results in a Disease Control Rate (DCR) in a population of patients who have received such treatment. In some embodiments, the HSV-1 is JP-OV-2. In some embodiments, the antibody that binds to PD-1 is cetrelimab. In some embodiments, the Disease Control Rate (DCR) is defined as the percentage of participants who have achieved complete response, partial response, and stable disease.
[0229] In some embodiments, provided herein is a method of treating an advanced solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual every two weeks (Q2W) intratumorally and administering an antibody that binds to PD- 1 about every four weeks (Q4W) intravenously, wherein such treatment results in Duration of Response (DOR) in a population of patients who have received such treatment thereby demonstrating the subject is treated. In some embodiments, the HSV-1 is JP-OV-2. In some embodiments, the antibody that binds to PD-1 is cetrelimab. In some embodiments, the Duration of Response (DOR) will be calculated among responders from the date of initial documentation of a response to the date of first documented evidence of relapse according to RECIST v1.1 , or death due to any cause, whichever occurs first. In some embodiments, the DOR in individuals with disease that have not relapsed and who are alive will be censored at the last disease evaluation before the start of any subsequent anticancer therapy. In some embodiments, such treatment increases the DOR in the individual.
[0230] In some embodiments, provided herein is a method of treating an advanced solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual every two weeks (Q2W) intratumorally and administering an antibody that binds to PD- 1 about every four weeks (Q4W) intravenously, wherein such treatment results in a Progression Free Survival (PFS) in a population of patients who have received such treatment. In some embodiments, the HSV-1 is JP-OV-2. In some embodiments, the antibody that binds to PD-1 is cetrelimab. In some embodiments, the Progression Free Survival (PFS) is defined as the time from treatment initiation until disease progression or worsening or death due to any cause. In some embodiments, such treatment increases the PFS in the individual.
[0231] In some embodiments, provided herein is a method of treating an advanced solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual every two weeks (Q2W) intratumorally and administering an antibody that binds to PD- 1 about every four weeks (Q4W) intravenously, wherein such treatment results in an Overall Survival (OS) in a population of patients who have received such treatment. In some embodiments, the HSV-1 is JP-OV-2. In some embodiments, the antibody that binds to PD-1 is cetrelimab. In some embodiments, the Overall Survival (OS) is defined as the time from treatment initiation until death due to any cause.
[0232] In some embodiments, provided herein is a method of treating an advanced solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual every two weeks (Q2W) intratumorally and administering an antibody that binds to PD- 1 about every four weeks (Q4W) intravenously, wherein such treatment is safe and well-tolerated. In some embodiments, the HSV-1 is JP-OV-2. In some embodiments, the antibody that binds to PD-1 is cetrelimab.
[0233] In some embodiments, also provided herein is a method of administering a safe and tolerable treatment for an advanced solid tumor in an individual comprising administering an oncolytic herpes simplex type 1 (HSV-1) virus to the individual every two weeks (Q2W) intratumorally and administering an antibody that binds to PD-1 about every four weeks (Q4W) intravenously. In some embodiments, the HSV-1 is JP-OV-2. In some embodiments, the antibody that binds to PD-1 is cetrelimab.
VIII. Pharmaceutical compositions
[0234] Further provided herein are pharmaceutical compositions comprising any of the oncolytic viruses (such as any of the oncolytic HSV) and PD-1 antibodies described herein, and optionally a pharmaceutically acceptable excipient, carrier, and/or stabilizer. Pharmaceutical compositions can be prepared by mixing a oncolytic virus (such as any of the oncolytic HSV) described herein having a desired degree of purity with pharmaceutically acceptable carriers, excipients, and/or stabilizers.
[0235] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, excipient, and/or stabilizer. Pharmaceutically acceptable, as used herein, includes any carrier, excipient, and/or stabilizer which does not interfere with the desired effectiveness or biological activity of the oncolytic virus (such as an oncolytic HSV) and/or that is not toxic to the subject to whom the pharmaceutical composition is, or is to be, administered. Pharmaceutically acceptable carriers, excipients, and stabilizers are known in the art, including in the Handbook of Pharmaceutical Excipients. Non-limiting examples of suitable carriers, excipients, and stabilizers include buffering agents preservatives, binders, compaction agents, lubricants, chelators, dispersion enhancers, disintegration agents, coloring agents, and the like..
IX. Kits and articles of manufacture
[0236] Further provided herein are kits and articles of manufacture comprising any of the oncolytic viruses (such as any of the oncolytic HSV) and PD-1 antibodies described herein, pharmaceutical compositions comprising any of the oncolytic viruses (such as any of the oncolytic HSV) and PD- 1 antibodies described herein, or isolated nucleic acids (such as the expression cassettes) described herein.
[0237] The kits of the present application are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Kits may optionally provide additional components such as buffers and interpretative information. The present application also provides articles of manufacture, which include vials, bottles, jars, flexible packaging, and the like.
[0238] The article of manufacture can comprise a container and a label or package insert on or associated with the container.
[0239] The kits or article of manufacture may include multiple unit doses of the pharmaceutical composition , packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies.
EXEMPLARY EMBODIMENTS
[0240] The present disclosure may be better understood with reference to the following exemplary embodiments.
[0241] Embodiment 1 is a method of treating an advanced solid tumor in an individual comprising administering to the individual an oncolytic herpes simplex type 1 (HSV-1) virus every two weeks (Q2W) intratumorally, wherein the HSV-1 comprises one or more expression cassettes comprising a polynucleotide encoding hFLT3L protein, a polynucleotide encoding a CTLA-4 binding protein, a polynucleotide encoding a CD40 agonist, and a polynucleotide encoding IL- 12.
[0242] Embodiment 2 is the method of embodiment 1, wherein the oncolytic HSV-1 virus comprises: a. a cassette integrated in one or both of the γ34.5 loci comprising in order, from upstream to downstream, a CMV promoter, a polynucleotide encoding hFLT3L, a P2A cleavage sequence, a polynucleotide encoding UL49.5, and a polyadenylation signal; and b. another cassette integrated in the US10-12 locus comprising in order, from upstream to downstream, a polynucleotide comprising a variant US11 gene encoding native US11 protein, an additional polynucleotide encoding native US11 protein, a polynucleotide encoding US 10 protein, a polyadenylation signal that is operably linked to the polynucleotide encoding the US 10 protein, a CMV promoter, a polynucleotide encoding a CTLA-4 binding protein, a polyadenylation signal that is operably linked to the polynucleotide encoding the CTLA-4 binding protein, a polyadenylation signal that is operably linked to a polynucleotide encoding a CD40 agonist, a polynucleotide encoding a CD40 agonist, an AoHVl promoter that controls expression of the CD40 agonist, an MMLV promoter that controls expression of an IL- 12, a polynucleotide encoding an IL- 12, and a polyadenylation signal that is operably linked to the polynucleotide encoding the IL- 12, wherein the polynucleotide for hFLT3L encodes the amino acid sequence set forth in SEQ ID NO: 71, the polynucleotide for UL49.5 encodes the amino acid sequence set forth in SEQ ID NO: 82, the polynucleotide for IL-12 encodes the amino acid sequence set forth in SEQ ID NO: 4 , the polynucleotide for CD40 agonist encodes the amino acid sequence set forth in SEQ ID NO: 25, and the polynucleotide for CTLA-4 binding protein encodes the amino acid sequence set forth in SEQ ID NO: 50, the polynucleotide for variant US11 gene comprises the polynucleotide sequence set forth in SEQ ID NO: 204, the additional polynucleotide encoding for US11 encodes the amino acid sequence set forth in SEQ ID NO: 80, and the polynucleotide for US 10 encodes the amino acid sequence set forth in SEQ ID NO: 90.
[0243] Embodiment 3 is the method of embodiment 1 or embodiment 2, wherein the oncolytic HSV-1 virus is JP-OV-2.
[0244] Embodiment 4 is the method of any one of embodiments 1-3, wherein the HSV-1 virus is administered to the individual as a monotherapy to treat the solid tumor.
[0245] Embodiment 5 is the method of any one of embodiments 1-4, wherein the HSV-1 virus is administered to the individual intratumorally in one or more lesions.
[0246] Embodiment 6 is the method of any one of embodiments 1-5, further comprising administering to the individual an antibody that binds to PD-1.
[0247] Embodiment 7 is the method of any of embodiments 1-6, wherein the individual does not have a central nervous system solid tumor.
[0248] Embodiment 8 is the method of any one of embodiments 1-7, wherein the individual has non-small cell lung cancer (NSCLC).
[0249] Embodiment 9 is the method of any one of embodiments 1-8, wherein the individual has relapsed or refractory metastatic NSCLC.
[0250] Embodiment 10 is the method of any one of embodiments 1-9, wherein the individual has received all available standard therapy and a solid tumor progressed. [0251] Embodiment 11 is the method of any one of embodiments 1-10, wherein the individual has stage IIIB-IV NSCLC.
[0252] Embodiment 12 is the method of any one of embodiments 1-11, wherein the individual was previously treated with (a) an anti-PD-1 or an anti-PD-L1 therapy; and (b) a platinum-based chemotherapy, either as combination or sequentially for a metastatic disease and the metastatic disease has progressed on or after therapy.
[0253] Embodiment 13 is the method of any one of embodiments 1-12, wherein the individual cannot tolerate or has previously refused the platinum based chemotherapy.
[0254] Embodiment 14 is the method of any one of embodiments 1-13, wherein the individual is unable to receive platinum-based chemotherapy and has progressed after the anti-PD-1 or the anti-PD-L1 therapy alone.
[0255] Embodiment 15 is the method of any one of embodiments 1-14 wherein the individual has a PD-L1 expression of less than 1% and has previously been treated with an anti-PD-1 therapy.
[0256] Embodiment 16 is the method of any one of embodiments 1-15 wherein the individual has a PD-L1 expression of greater than 50% and has not received a prior systemic therapy for the metastatic disease.
[0257] Embodiment 17 is the method of any one of embodiments 1-16, wherein the HSV-1 virus is administered to the individual at a dose of about 105 about, 106, about 107, or about 108 PFU/mL.
[0258] Embodiment 18 is the method of any one of embodiments 1-17, wherein the HSV-1 virus is administered to the individual at a dose of about 105 PFU/mL.
[0259] Embodiment 19 is the method of any one of embodiments 1-18, wherein the HSV-1 virus is administered to the individual at a dose of about 106 PFU/mL.
[0260] Embodiment 20 is the method of any one of embodiments 1-19, wherein the HSV-1 virus is administered to the individual at a dose of about 107 PFU/mL.
[0261] Embodiment 21 is the method of any one of embodiments 1-20, wherein the HSV-1 virus is administered to the individual at a dose of about 108 PFU/mL.
[0262] Embodiment 22 is the method of any one of embodiments 1-21, wherein the volume of the HSV-1 injected per lesion is determined based on the longest diameter of the lesion. [0263] Embodiment 23 is the method of any one of embodiments 1-22, wherein the HSV-1 virus is administered to the individual in a maximum volume of 10 ml.
[0264] Embodiment 24 is the method of any one of embodiments 1-23, wherein the HSV-1 virus is injected to the individual into a cutaneous, subcutaneous, or nodal lesion.
[0265] Embodiment 25 is the method of embodiment 24, wherein the HSV-1 virus is administered to the cutaneous, subcutaneous, or nodal lesion in a superficial approach with injection under direct visualization and palpation.
[0266] Embodiment 26 is the method of any one of embodiments 1-25, wherein the HSV-1 virus is injected to the individual into a transthoracically accessible lung lesion, a transabdominally accessible liver lesion, and/or a subcutaneous soft tissue lesion.
[0267] Embodiment 27 is the method of embodiment 26, wherein the HSV-1 virus is administered to the transthoracically accessible lung lesion, the transabdominally accessible liver lesion, and/or the subcutaneous soft tissue lesion in a percutaneous administration approach via imaging guidance.
[0268] Embodiment 28 is the method of any one of embodiments 1-27, wherein the HSV-1 virus is administered to a lung lesion or a lymph node accessible via a bronchoscope.
[0269] Embodiment 29 is the method of any one of embodiments 1-28, wherein the individual receives a maximum of 8 doses of the HSV-1 virus.
[0270] Embodiment 30 is the method of any one of embodiments 6-29, wherein the antibody that specifically binds to PD-1 comprises a heavy chain variable region (VH) comprising a heavy chain complementarity-determining region (CDRH1) comprising the amino acid sequence set forth in SEQ ID NO: 401, a CDRH2 comprising the amino acid sequence set forth in SEQ ID NO: 402, a CDRH3 comprising the amino acid sequence set forth in SEQ ID NO: 403; and a light chain variable region (VL) comprising a light chain complementarity-determining region (CDRL1) comprising the amino acid sequence set forth in SEQ ID NO: 405, a CDRL2 comprising the amino acid sequence set forth in SEQ ID NO: 406, and a CDRL3 comprising the amino acid sequence set forth in SEQ ID NO: 407.
[0271] Embodiment 31 is the method of embodiment 30, wherein the antibody that specifically binds to PD-1 comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 408 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 409. [0272] Embodiment 32 is the method of embodiment 31, wherein the antibody that specifically binds to PD-1 comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 400 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 404. [0273] Embodiment 33 is the method of any one of embodiments 6-32, wherein the antibody that binds to PD-1 is cetrelimab.
[0274] Embodiment 34 is the method of any one of embodiments 6-33, wherein the antibody that binds to PD-1 is administered to the individual at a dose of about 480 mg.
[0275] Embodiment 35 is the method of any one of embodiments 6-34, wherein the antibody that binds to PD-1 is administered to the individual every four weeks (Q4W).
[0276] Embodiment 36 is the method of any one of embodiments 6-35 wherein the antibody that binds to PD-1 is administered to the individual every four weeks (Q4W) for at least about 15 weeks.
[0277] Embodiment 37 is the method of embodiment 36, wherein the antibody that binds to PD- 1 is administered to the individual four weeks (Q4W) for at most about 2 years.
[0278] Embodiment 38 is the method of any one of embodiments 6-37, wherein the antibody that binds to PD-1 is administered intravenously.
[0279] Embodiment 39 is the method of any one of embodiments 6-38, wherein the antibody that binds to PD-1 is administered to the individual after the HSV-1 virus.
[0280] Embodiment 40 is the method of any one of embodiments 6-39, wherein the antibody that binds to PD-1 is administered to the individual starting in week three of treatment, wherein week one of treatment is the first administration of the HSV-1 virus.
[0281] Embodiment 41 is the method of any one of embodiments 6-40, wherein the antibody that binds to PD-1 is administered to the individual starting in week one of treatment, wherein week one of treatment is the first administration of the HSV-1 virus.
[0282] Embodiment 42 is the method of any one of embodiments 1-41, wherein the size of the solid tumor is reduced.
[0283] Embodiment 43 is the method of any one of embodiments 1-42, wherein the individual has more than one solid tumor.
[0284] Embodiment 44 is the method of embodiment 43, wherein the HSV-1 is administered to at least one of the solid tumors. [0285] Embodiment 44 is the method of embodiment 44, wherein the size of a solid tumor other than the at least one solid tumor where the HSV-1 is administered decreases in size following treatment.
[0286] Emdodiment 46 is a method of optimizing a dosing regimen of JP-OV-2 for treating an advanced solid tumor in an individual, comprising: a. evaluating the first three dose escalation levels of JP-OV-2 in a bi-weekly (Q2W) schedule; and b. implementing one or more alternative dosing regimens for JP-OV-2; wherein said alternative dosing regimens involve a dose intensification.
[0287] Embodiment 47 is the method of embodiment 46, wherein the individual receives JP-OV- 2 in combination with cetrelimab starting from the first dose of JP-OV-2.
[0288] Embodiment 48 the method of embodiment 46 or 47, wherein the dose intensification comprises: a. administering JP-OV-2 on Days 1 and 8 of a 28-day cycle of cetrelimab as a first intensified dose; b. evaluating the safety and efficacy of the first intensified dose; and c. administering a second intensified dose of JP-OV-2 based on the evaluation of (b). [0289] Embodiment 49 is the method of any one of embodiments 46-48, further comprising administering up to an additional 8 doses of JP-OV-2 after the individual has received 8 doses from an initial dosing regimen.
SEQUENCES
Table 6: CD40 Agonist Payload Amino Acid Sequences.
Table 7: CTLA-4 Antagonist Pay load Amino Acid Sequences.
Table 8: Human FLT3L Payload Amino Acid Sequences.
Table 9: Immune Stealth Protein Amino Acid Sequences.
Table 10: Additional amino acid sequences.
Table 11: γ34,5 Locus Cassete Sequences.
Table 12: US10-12 Locus Cassete Sequences.
Table 13: Additional Nucleotide Sequences.
Table 14: Cetrelimab Sequences.
EXAMPLES
[0290] The presently disclosed subject matter will be better understood by reference to the following Examples, which are provided as exemplary of the invention, and not by way of limitation.
EXAMPLE 1 : ONCOLYTIC HSV-1 VIRUS IN COMBINATION WITH AN ANTI-PD1 ANTIBODY IN AN MC 38 5 AG TUMOR MODEL. [0291] This example describes an oncolytic HSV-1 virus in combination with an anti-PD-1 antibody. The genome architecture of the mouse surrogate HSV-1 virus, mJP-OV-2, and the human HSV-1 virus, JP-OV-2, are provided in FIGs. 1A-1B. Briefly, the mJP-OV-2 virus was engineered to express several immunomodulatory payloads including an anti-CTLA-4 antagonist (mαCTLA-4), a CD40 agonist (mCD40ag), IL- 12 (mscIL-12), and hFLT3L. The virus was also engineered to express and UL49.5, codon-optimized US 11 (hCoUS11) using the US 12 immediate early promoter, and endogenous US 11 using late US 11 promoter. The mouse surrogate HSV-1 virus mJP-OV-2 was engineered with 4 immunomodulatory payloads: hFLT3L, mCD40 ag, αmCTLA4, and mscIL12.8 known to activate all stages of adaptive immunity. hFLT3L was retained in mJP-OV-2, as this payload is cross-reactive between mouse and human.
[0292] FIG. 2A depicts a diagram of the experimental workflow to assess treatment with an oncolytic HSV-1 virus in combination with an anti-PD-1 antibody. The general experimental design was to implant tumor cells bilaterally, followed by intratumoral treatment of established tumors on the right flank. Treated and untreated tumors were monitored to assess if the treatment inhibited growth of the primary tumor as well as an abscopal effect on growth of the secondary untreated tumor, which would indicate that the mice had developed an adaptive immune response to tumor antigens.
[0293] Female C57BL/6 mice were obtained from Charles River Laboratories and were enrolled when they were approximately 8 weeks of age with an average body weight of 18 to 20 g. The mouse syngeneic cancer cell line, MC-38-5AG, was obtained from Janssen R&D, Spring House, PA, and was grown in complete culture medium. MC-38-5AG cells were harvested during exponential growth on Day 0, using TrypLE. Cells were washed twice in cold DPBS and resuspended in cold DPBS at a concentration of 5× 106 cells/mL. Mice were implanted bilaterally in each flank by SC injection with 0.1 mL of the cell suspension (ie, 5×105 cells in each flank). In all studies, Day 0 was the day of tumor cell implantation and study initiation. Cells were implanted SC in both the left and right flanks, just below the ribcage. Mice were randomized by the Multi Task method in Studylog software (Studylog Systems, Version 4.3) according to tumor volume (TV), using the right side as the primary tumor, followed by the left side as the secondary tumor, such that the p value was as close to 1 as possible and the percent difference and standard deviations were similar among the groups within each cohort. All intratumoral injections were in 0.05 mL and were performed on the right tumor. The vehicle control group was dosed with BeneVir formulation buffer in each study.
[0294] Mice were randomized into groups of 10 animals each, 7 days post MC-38-5AG cell implantation according to tumor volume (TV), with mean right and left TV of 26 mm3 and 24 mm3, respectively. On Days 8, 11, and 14, mice received intratumoral injections of vehicle or mJP- OV-2. On Days 8, 12, 15, and 19, mice received intraperitoneal (IP) injections of anti-PD-1 antibody RMP1 14 according to the study design.
[0295] At study end, as determined by twice the median survival day of the vehicle control group, naive mice (n=10) were challenged, and any cured mice with CRs were rechallenged with 5× 105 MC 38 5 AG cells implanted in the left flank. All mice were monitored until a median survival day was assessed from the naive group, at which point a study end date was determined for the rechallenge. A summary of the study design described above is provided in Table 15.
[0296] Significant antitumor effect was observed for the tumors treated with the oncolytic HSV-1 virus with or without anti-PD-1 antibody RMP1 14, or with anti-PD-1 alone, as compared to vehicle control mice over time to Day 29 post tumor implantation (p≤0.001 ; FIG. 2B). In addition, a significant abscopal effect on the untreated tumors was observed for mice treated with the oncolytic HSV-1 virus alone or in combination with anti-PD-1 (p≤0.001). Survival analysis of these groups demonstrated that the oncolytic HSV-1 virus, with or without anti-PD-1, significantly enhanced overall survival (p≤0.001; FIG. 2C). On Day 61, complete responses (CRs) were observed for 5 of 10 mice treated with the oncolytic HSV-1 virus and 10 of 10 mice treated with the oncolytic HSV-1 virus + anti-PD-1. Anti-PD-1 also significantly increased survival (p≤0.001), but no CRs were observed in this group on Day 61. These results indicate that the combination of the oncolytic HSV-1 virus + anti-PD-1 was more effective than the single agents to inhibit tumor growth and prolong survival.
[0297] These results suggest that activity of the anti-PD-1 therapy to block the interaction of PD- 1 and programmed death-ligand 1 (PDL1), enhances the antitumor activity of the oncolytic HSV- 1 virus by abolishing the inhibition of CD8+ T cells. Further, the combination of the oncolytic HSV-1 virus and PD-1 antibody was effective for treating solid tumors and the two agents act synergistically to provide a stronger anti-tumor effect than either agent individually. Further, the tumor reduction observed on the untreated tumor demonstrate that treatment with the oncolytic HSV-1 virus and a PD-1 antibody resulted in a significant abscopal effect.
EXAMPLE 2: PHASE 1 STUDY OF INTRATUMORAL ADMINISTRATION OF JP-OV-2, AN ONCOLYTIC VIRUS, AS MONOTHERAPY AND IN COMBINATION FOR ADVANCED SOLID TUMORS
[0298] This example describes a Phase 1 study of intratumoral administration of JP-OV-2, an oncolytic virus, as monotherapy and in combination with a PD-1 antibody, cetrelimab, for advanced solid tumors.
STUDY OVERVIEW
[0299] The study design is illustrated in FIGS. 3A-3C.
[0300] JP-OV-2 is a next-generation recombinant HSV Type-l-based oncolytic virus (OV) that has been modified to 1) reduce innate and adaptive antiviral host responses that shut down viral replication to allow greater lysis of target tumor cells and 2) to enhance all stages of the adaptive immune response to the cancer cells through expression of multiple synergistic immune stimulatory payloads. These features are postulated to result not only in sustained anti-tumor activity within injected lesions, but also to stimulate anti -tumor activity in distant uninjected lesions.
[0301] Cetrelimab is a fully human IgG4K monoclonal antibody that binds to PD-1. Cetrelimab comprises a heavy chain and a light chain, wherein the heavy chain of the cetrelimab comprises a heavy chain variable region (VH) comprising a CDR-H1, CDR-H2, and CDR-H3 of a VH comprising the amino acid sequence set forth in SEQ ID NO:400, and the light chain of the cetrelimab comprises a light chain variable region (VL) comprising a CDR-L1, CDR-L2, and CDR-L3 of a VL comprising the amino acid sequence set forth in SEQ ID NO:404. Cetrelimab comprises a VH comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 401, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 402, a CDR- H3 comprising the amino acid sequence set forth in SEQ ID NO: 403; and/or a VL comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 405, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 406, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 407.
[0302] This study evaluates JP-OV-2 administered via intratumoral injection as a monotherapy and in combination with a PD-1 antibody.
[0303] Initially, approximately 96 participants are planned to be treated. In Part 2, the first expansion cohort will consist of participants with relapsed (Cohort A) and treatment-naive (Cohort B) metastatic NSCLC.
[0304]
Part 1, Dose Escalation
[0305] JP-OV-2 is given via intratumoral injection Q2W. Dose Escalation of JP-OV-2 begins at a starting dose of 106PFU/mL (number of viral plaque-forming units per mL of solution) with the number of mLs administered dependent on the size of the tumor. At each treatment visit, 1 or more lesions may be injected, and the maximum volume administered across all injected lesions is 10 mL. The first dose of JP-OV-2 is administered to each participant as a monotherapy and then in combination with cetrelimab beginning with the second dose of JP-OV-2. The first participant treated at a dose level is evaluated for at least 5 days prior to treating subsequent participants. Cetrelimab is administered at a dose of 480 mg IV once every 4 weeks (Q4W). Once a dose level of JP-OV-2 is determined by the study evaluation team (SET) to be safe as a monotherapy, additional participants may be enrolled to the next higher dose level of JP-OV-2 monotherapy as guided by the BOIN design. Combination of JP-OV-2 at the higher dose level with cetrelimab will not begin until the SET confirms the safety of the JP-OV-2 and cetrelimab combination at the previous dose level during a 14-day combination DLT observation period. A dosing table (Table 16) is provided below.
Table 16: Dosing Table for JP-OV-2
[0306] Core needle biopsies may be obtained from on-treatment tumor samples during Part 1 to assess changes in the tumor microenvironment. At least 1 injected and at least 1 uninjected lesion prior to the fourth dose of JP-OV-2 will be obtained. The injected lesion should be the same as the one biopsied at baseline.
[0307] There are 2 dose limiting toxicity (DLT) evaluation periods: a monotherapy DLT evaluation period and a combination DLT evaluation period.
[0308] The monotherapy DLT evaluation period is defined by the scheduled interval between IT dosing, corresponding to 14 days after receiving the first injection with JP-OV-2 and the combination DLT evaluation period as 14 days after initiating combination therapy with JP-OV-2 and cetrelimab.
[0309] One or more safe and feasible dose(s) for JP-OV-2 as monotherapy and in combination with cetrelimab, will be further investigated in Part 2 after review of all available pharmacokinetic, pharmacodynamic, safety, and preliminary anti-tumor activity data.
[0310] Once the first 3 dose levels have been evaluated, additional dose levels may be enrolled per SET decision, to evaluate alternative JP-OV-2 dosing frequencies or ratios of tumor volume to injected JP-OV-2 volume.
Alternative Dosing Regimens and JP-OV-2 Dosing Volumes per Lesion Size
[0311] Once the first 3 Part 1 dose escalation levels have been evaluated using Q2W schedule for JP-OV-2 and upon approval by the SET, alternative JP-OV-2 dosing regimens (dose intensification) may be implemented in Part 1 , if available safety, efficacy, and translational data suggest further optimization of schedule is feasible. If these alternative (intensified) schedules of activities are found to be safe and feasible in Part 1 , they may be implemented in Part 2 of the study.
[0312] In these alternative JP-OV-2 dosing regimens (dose intensification), treatment will be initiated and continued with JP-OV-2 in combination with cetrelimab so that participants will receive combination treatment starting with the first dose. As in the Q2W schedule, up to 8 treatments with JP-OV-2 may be given. Additional treatments, up to 8 more doses, may be given with sponsor approval on a case-by-case basis and upon satisfying defined criteria. [0313] The first alternative (intensified) dosing regimen (Table 17) will evaluate JP-OV-2 administration on Days 1 and 8 of each 28-day cycle of cetrelimab. If this regimen is deemed safe and feasible by the SET, a second intensified regimen may be evaluated (both doses within first 7 days of each 28-day cycle of cetrelimab) based upon safety, and PK data, to optimize intratumoral payload effect (Table 18).
[0314] In addition to evaluating alternative (intensified) dose regimens, the SET may decide to evaluate higher ratios of JP-OV-2 per tumor volume only after the first 3 dose levels have been evaluated and the SET have agreed that it is safe to do so. Higher ratios of injected JP-OV-2 to tumor volume will be achieved by limiting the largest injected lesion diameter to 4 cm (vs >5 cm per Table 20) to receive the full 10 mL target JP-OV-2 injection. The higher injected JP-OV-2 volumes per lesion size are provided in Table 19. These injection volumes may be evaluated on Q2W schedule or alternative schedules, provided the schedule has been used with the dose volumes defined in Table 20.
Table 17: Schedule of Activities for Alternative (Intensified) JP-OV-2 Dosing Schedule #1
Table 18: Schedule of Activities for Alternative (Intensified) JP-OV-2 Dosing Schedule #2
Table 19: Alternative Injection Volume per Lesion Based on Lesion Size
Note: The volume listed in the table should be the full amount prescribed for lesions in each size category, “up to” denotes the possibility that the full amount may not be feasible to administer to the lesion. This may occur, for example, if the lesion has decreased in size since the last disease assessment or if the pressure required to administer the study treatment becomes prohibitive in the opinion of the treating physician.
“Large or spherical superficial lesions may be injected using the visceral injection volumes.
Part 2, Dose Expansion
[0315] Part 2 of the study will further evaluate the safety and tolerability of JP-OV-2 (used at the doses and regimens identified in Part 1) in combination with a PD-1 antibody and will also assess the preliminary efficacy of the treatment regimen. Cohorts A and B will evaluate treatment with JP-OV-2 in combination with cetrelimab at the dose identified in Part 1 in participants with metastatic NSCLC who have progressed after treatment with anti-PD-1 and chemotherapy. In this cohort, treatment is initiated and continued with combination of JP-OV-2 in combination with cetrelimab so that participants will receive combination treatment starting with the first dose. For Part 2, Cohort B will be initiated after review of safety data from the first 3 participants in Cohort A treated with the combination treatment for at least 14 days. Additionally, treatment for the first 3 participants receiving endobronchial administration of JP-OV-2 into the lung-lesions in either cohort will be staggered by at least 5 days each. Cohort A of Part 2 will consist of participants with advanced metastatic NSCLC who have progressed after treatment with anti-PD-1 and chemotherapy and have a PD-L1<1% tumor proportion score. Cohort B of Part 2 will consist of participants with advanced metastatic NSCLC who have never been treated for their metastatic disease and a PD-L1>50% tumor proportion score. Up to 30 participants may be treated in each Cohort A and B.
Administration of JP-OV-2
[0316] JP-OV-2 is administered Q2W via intratumoral injection up to a maximum volume of 10 mL divided within the selected lesions. Based upon evolving data, the SET may decide to investigate additional dosing regimens as described previously. Although doses may be skipped based on clinical feasibility and medical condition of the participant, every effort should be made to ensure that the participants receive all scheduled doses prior to the first disease assessment to maximize probability of clinical benefit. Injections may be performed by 3 approaches depending on locations of lesions and part of study enrollment. Different approaches may be utilized at the same or different visits depending on the location of all injectable lesions and clinical feasibility. [0317] In Parts 1 and 2: JP-OV-2 can be administered via a superficial approach with injection under direct visualization/palpation into a cutaneous, subcutaneous, or nodal lesion (ultrasound guidance optional to facilitate/monitor but not required);
[0318] In Parts 1 and 2: JP-OV-2 can be administered via a percutaneous approach for lesions accessible via imaging guidance (such as transthoracically accessible lung lesions; transabdominally accessible liver lesions, subcutaneous soft tissue lesions etc.); and,
[0319] In Part 2: JP-OV-2 can be administered via an endobronchial approach for lung lesions and lymph nodes accessible via a bronchoscope.
[0320] At each treatment visit, 1 or more lesion(s) may be injected provided all injections are performed on the same day. For injections in the lung, only lesions in 1 hemithorax (1 lung) can be injected per treatment visit.
[0321] If administration of cetrelimab is not possible on the day of JP-OV-2 administration, then cetrelimab may be administered on the next day.
[0322] Treatment may be continued for up to a maximum of 8 doses of JP-OV-2 and 2 years with cetrelimab unless discontinuation criteria are reached earlier (confirmed radiographic disease progression, unequivocal clinical disease progression, unacceptable toxicity, withdrawal of consent, investigator decision, the participant becomes pregnant, or the participant is lost to follow- up). Treatment may be continued after confirmed radiographic disease progression if deemed by the investigator to be in the best interest of the participant and after obtaining approval from the physicians involved in participant’s treatment and from the sponsor medical monitor. Continuation or reinitiation of JP-OV-2 up to an additional 8 doses may be permitted during cetrelimab treatment after consultation with the sponsor medical monitor.
JP-OV-2 Injection Volume
[0323] Injection volume for each lesion is based on the longest diameter of the lesion (including lymph nodes) per the most recent disease evaluation radiographic scan prior to scheduled treatment and following the doses provided in Table 17. The maximum injection volume per administration/treatment cycle is 10 mL. This volume may be divided into aliquots and injected into multiple tumor lesions via the appropriate injection route (superficial, percutaneous, or endobronchial). At each administration, as many lesions as possible should be injected keeping the total injected volume as close to 10 mL as feasible. For injections into visceral lesions, no more than 3 lesions within a single solid organ should be injected at a single administration visit unless discussed with the sponsor. During the injection procedure, if there is a need to change the dose volume based on intraprocedural imaging (CT or CBCT), the change must be documented.
[0324] Once the injection volumes listed in Table 20 are evaluated in the first 3 dose levels of Part 1 and following SET approval, increased dose administration using higher injection volumes per lesion size may be evaluated in separate dose cohorts as described previously.
Table 20: Injection Volume Per Lesion Based on Lesion Size
Injectable Lesion Selection
[0325] Participants may receive injections in more than one organ/site of disease and the intratumoral injection procedure is determined by the lesion type and location. At each treatment visit, one or more lesions may be injected, and the maximum total JP-OV-2 dose volume injected across all lesions is 10 mL, with injection volume per lesion guided by Table 17. For injections in the lung, only lesions in one hemithorax (one lung) can be injected per administration.
[0326] The number of lesions and organ sites to be injected is determined based on the number and size of accessible lesions to achieve a total dose volume of 10 mL injected in all lesions at each JP-OV-2 treatment visit.
[0327] For participants in Part 2, at least 1 lesion selected as an uninjectable target lesion for RECIST v1.1 assessment should not be injected, unless all other lesions become uninjectable. In this case, sponsor must be consulted prior to injecting the remaining uninjected target lesion.
Guidelines for Lesion Prioritization for Injection
[0328] It is recommended that each lesion be injected with the maximum amount recommended per Table 17 before moving to the next lesion, subject to tumor specific limitations. Clusters of lesions may be considered as a single lesion for injection. [0329] Lesions for initial injections should be selected as to increase the feasibility for biweekly injection, at least through the first disease assessment, to maximize possibility of achieving clinical benefit. For the first administration visit, it is recommended to inject the largest injectable lesion that is clinically appropriate. For Part 2, the largest injectable lung lesion should be prioritized for injection via endobronchial or transthoracic approach if feasible. Primary lung lesion and affected measurable lymph nodes should be injected next. Inject any remaining injectable lesions based on the lesion size until the maximum volume of 10 mL has been injected or no injectable lesions remain.
Administration
[0330] A superficial lesion is a cutaneous, subcutaneous, or nodal lesion that can be accessed in the clinic under direct visualization/palpation (ultrasound guidance optional to facilitate/monitor but not required).
[0331] Visceral lesions or deep nodal lesions (collectively referred to as “visceral”) require imaging guidance (ultrasound, CBCT, or CT) for injection. Visceral lesions should be safe to inject in the opinion of the investigator (eg, avoiding lesions with major airway or blood vessel invasion). Lesions in locations with a history of poor wound healing should be avoided. Lesions in previously irradiated fields or other risk factors for poor wound healing (eg, poor vascularization) should be discussed with sponsor’s medical monitor prior to administration. For injections into visceral lesions, no more than 3 lesions within a single solid organ should be injected at a single administration visit unless discussed with the sponsor.
[0332] In Part 2, injections into the lung may involve endobronchial injection via a bronchoscope or percutaneous image-guided injections for lesions that are not accessible by a bronchoscope, based on the clinical decision of the performing physician.
STUDY POPULATION
Inclusion Criteria
[0333] Each potential participant must satisfy all of the following criteria to be enrolled in the study: [0334] 1. Be >18 years of age (or the legal age of majority in the jurisdiction in which the study is taking place, whichever is greater) at the time of informed consent.
[0335] 2.1. Part 1: Individuals with a diagnosis of advanced or metastatic solid tumor (except tumors of the CNS), who have previously received available standard therapy and progressed, or cannot tolerate standard therapy, or for whom there is no standard of care per regional guidelines. Individuals who have previously refused standard therapy in consultation with their health care professional may participate as long as documentation of the medical endorsement for their decision is provided.
[0336] 2.2. Part 2: Individuals with histologically or cytologically confirmed, metastatic or locally advanced NSCLC, who are not candidates for curative therapy. Disease must be confirmed to lack EGFR or Alk-based driver mutations at the time of diagnosis. Additional cohort-specific criteria are outlined below:
Cohort A:
• have a PD-L1<1% tumor expression based on the results of local testing performed in a CLIA-certified laboratory at the time of initial diagnosis.
• have been previously treated with anti-PD-1/PD-L1 therapy and platinum-based chemotherapy, either as combination or sequentially in the metastatic setting and have progressed on or after therapy. Individuals who cannot tolerate platinum-based chemotherapy are eligible to enroll based on progression after anti-PD-1/PD-L1 therapy alone. Individuals who have previously refused platinum-based chemotherapy in consultation with their health care professional may participate as long as documentation of the medical endorsement for their decision is provided.
• Cohort B:
• have a PD-L1>50% tumor expression local testing performed in a CLIA-certified laboratory at the time of initial diagnosis.
• have not received previous systemic therapy for metastatic disease.
• have never received anti-PD-(L)1 or anti-CTLA4 therapy.
• NOTE: participants who have received 1 dose of anti-PD-(L)1 treatment (first dose of standard of care) may be eligible as long as they (a) have not had any disease assessment scans after anti-PD-(L)1 administration AND (b) are able to complete all screening assessments and begin study treatment at the time of the next scheduled anti-PD-(L)1 administration (+/- 4 days).
[0337] 3. Have at least 1 injectable tumor >1 cm in longest diameter (or shortest diameter for lymph nodes) or injectable tumors that in aggregate are >1 cm in the longest diameter, which is/are also measurable by RECIST v1.1. Injectable lesion considerations (eg, injectable lesion must not be invading or in close proximity to major or large blood vessels or invading major airways). Eligibility for intratumoral treatment must be reviewed with all physicians to be involved in the participant’s treatment.
[0338] NOTE: for participants enrolled in alternative dose volume levels, an additional requirement is the presence of at least 1 injectable tumor measuring ≤4 cm in longest diameter.
[0339] 4. Part 2: Have at least 1 measurable lesion that will not be injected during the study unless approved by the Sponsor.
[0340] 5. ECOG performance status of Grade 0 or 1.
[0341] 6. Hematology laboratory parameters within the following range, independent of transfusion within 7 days, or granulocyte colony stimulating-factor within 2 weeks prior to first dose of study treatment: Hemoglobin >9.0 g/dL, Absolute neutrophil count >1.0 x 103/μL, Platelet count ≥75x 103/μL.
[0342] 7. Adequate liver function: Participants with no underlying hepatic metastases are eligible if they have: AST <3 x ULN, ALT <3 x ULN, and Total bilirubin <1.5 x ULN (isolated total bilirubin >1.5 x ULN with conjugated [direct] bilirubin <1.5 x ULN is allowed for those participants with known congenital nonhemolytic hyperbilirubinemias). Participants with known hepatic metastases are eligible if they have: AST <5 x ULN, ALT <5 x ULN, and Total bilirubin <3 x ULN (isolated total bilirubin ≥3 x ULN with conjugated [direct] bilirubin <1.5 x ULN is allowed for those participants with known congenital nonhemolytic hyperbilirubinemias).
[0343] 8. Adequate renal function with creatine clearance >50 mL/min calculated using Cockcroft-Gault formula.
[0344] 9. A participant must not be of childbearing potential. Eligible participants include: (a) female participants not of childbearing potential.
[0345] OR:
[0346] male participants who have documented evidence of vasectomy or azoospermia due to other reasons.. [0347] 10. A participant of childbearing potential must have a negative highly sensitive serum (eg, β-hCG) pregnancy test at screening and within 72 hours of the first dose of study treatment and must agree to further serum or urine pregnancy tests during the study.
[0348] 11. A participant using oral contraceptives must use an additional barrier contraceptive method.
[0349] 12. A participant must agree not to be pregnant, breastfeeding, or planning to become pregnant while enrolled in this study or within 5 months after the last dose of study treatment.
[0350] 13. A participant must agree not to donate gametes (ie, eggs or sperm) or freeze for future use for the purposes of assisted reproduction during the study and for a period of 5 months after receiving the last dose of study treatment. Participants should consider preservation of gametes prior to study treatment as anticancer treatments may impair fertility.
[0351] 14. A participant must wear a condom when engaging in any activity that allows for passage of ejaculate to another person during the study and for 5 months after receiving the last dose of study treatment. A participant who is vasectomized must still use a condom (with or without spermicide).
[0352] 15. Thyroid function laboratory values within normal range. Note: If thyroid stimulating hormone (TSH) is not within normal limits, the subject may still be eligible if triiodothyronine (T3) (total or free) and free thyroxine (T4) are within normal limits.
Exclusion Criteria
[0353] Any potential participant who meets any of the following criteria is excluded from participating in the study:
[0354] 1. Active disease involvement of the central nervous system (eg, primary central nervous system tumors, metastases, leptomeningeal disease) with the exception of the brain metastases that are (1) definitively, locally treated AND (2) clinically stable and asymptomatic for >2 weeks AND (3) who are off steroids or receiving low dose corticosteroid treatment (≤10 mg prednisone or equivalent) for at least 2 weeks prior to start of study treatment.
[0355] 2. Prior or concurrent second malignancy (other than the disease under study) that due to natural history or treatment is likely to interfere with any study endpoints of safety or the efficacy of the study treatment(s). Consultation with sponsor is required in such cases prior to enrollment. [0356] 3. Toxicity related to prior anticancer therapy that has not returned to Grade ≤1 or baseline levels (except for alopecia, vitiligo, Grade ≤2 peripheral neuropathy, and endocrinopathies that are stable on hormone replacement, which may be Grade 2).
[0357] 4. History of irAEs from prior anticancer therapy leading to treatment discontinuation, except for endocrinopathies that are stable on hormone replacement.
[0358] 5. Prior history of, or active, significant herpetic infections (eg, herpetic keratitis or encephalitis) or active herpetic infections that require ongoing systemic anti-viral therapy.
[0359] 6. Active infection or condition that requires treatment with systemic anti-infective agents (eg, antibiotics, antifungals, or antivirals) within 7 days prior to the first dose of study treatment or chronic use of anti-infective agents. Intermittent use of topical anti-infective agents is not exclusionary.
[0360] 7. Active autoimmune disease that requires systemic immunosuppressive medications (eg, chronic corticosteroid, methotrexate, or tacrolimus) within the 12 months prior to signing consent.
[0361] 8. Any other inherited or acquired conditions that render the participant significantly immunocompromised.
[0362] 9. History of non-infectious pneumonitis or interstitial lung disease that required systemic treatment with corticosteroids, or requirement for continuous supplemental oxygen use to maintain adequate oxygenation.
[0363] 10. History of solid organ or hematologic stem cell transplantation.
[0364] 11. Known positive test result for HIV or other immunodeficiency syndrome.
[0365] 12. Active bleeding diathesis or requirement for therapeutic anticoagulation that cannot be interrupted or altered for procedures.
[0366] 13. Venous thromboembolic events (eg, pulmonary embolism) within 1 month prior to the first dose of study treatment; uncomplicated (Grade ≤2) deep vein thrombosis is not considered exclusionary.
[0367] 14. Clinically significant cardiovascular disease, including any of the following within 6 months prior to signature of informed consent: Myocardial infarction, severe or unstable angina, or coronary artery bypass surgery, clinically significant arrhythmias (eg, ventricular arrhythmias or atrial fibrillation with uncontrolled heart rate), congestive heart failure (NYHA class III/V), cerebrovascular accident, transient ischemic attack, or other arterial thromboembolic event, or Myocarditis.
[0368] 15. Known allergies, hypersensitivity, or intolerance to JP-OV-2 or its excipients.
[0369] 16. Known allergies, hypersensitivity, or intolerance to cetrelimab or its excipients.
[0370] 17. Known allergy to anti-HSV therapies (eg, acyclovir, valacyclovir, or famciclovir).
[0371] 18. Had major surgery (eg, requiring general anesthesia) within 2 weeks before the first dose of study treatment, or will not have fully recovered from surgery prior to the first dose.
Participants with recent or planned surgical procedures utilizing only local anesthesia may participate.
[0372] 19. Part 1 and Part 2 Cohort A: Prior treatment with an HSV-based oncolytic virus for the treatment of metastatic disease.
[0373] Part 2 Cohort B: Any prior treatment for metastatic disease with the exception of a single dose of anti-PD(L)-1 agent administered prior to the first dose of study treatment (see Inclusion Criterion 2). Chemotherapy for non-metastatic NSCLC is allowed as long as metastatic disease did not develop within one year following this treatment. Radiation therapy or surgery for non- metastatic disease are allowed as long as the windows specified in other exclusion criteria are respected.
[0374] 20. Part 1 and Part 2 Cohort A: Prior anti-PD(L)-l or anti-CTLA-4 therapy within 4 weeks or other anti cancer therapy within 14 days before the first dose of study treatment.
[0375] 21. Radiation therapy within 7 days before the first dose of study treatment.
[0376] 22. Received immunosuppressive doses of systemic medications, such as corticosteroids (doses >10 mg/day prednisone or equivalent) within 7 days prior to the first dose of study treatment. A single course of corticosteroids is permitted as allergy prophylaxis for imaging contrast.
[0377] 23. Received or plans to receive any live vaccine within 28 days before the first dose of study treatment. Non-live or non-replicating vaccines approved (eg, influenza) or authorized for emergency use (eg, COVID-19) by local health authorities are allowed.
[0378] 24. Received an investigational pharmaceutical intervention or used an invasive investigational medical device within 30 days before the planned first dose of study treatment.
[0379] 25. Active hepatitis of infectious origin. Seropositive for hepatitis B: defined by a positive test for hepatitis B surface antigen [HbsAg], Participants with resolved infection (ie, participants who are HbsAg negative with positive antibodies to total hepatitis B core antigen [anti- HBc] must be screened using real-time polymerase chain reaction (RT-PCR) measurement of hepatitis B virus (HBV) DNA levels. Those who are RT-PCR positive are excluded. Participants with serologic findings suggestive of HBV vaccination (anti-HBs positivity as the only serologic marker) AND a known history of prior HBV vaccination, do not need to be tested for HBV DNA by RT-PCR. (see Appendix 9, Hepatitis B Virus Testing). Known active hepatitis C infection or positive serologic testing for hepatitis C virus (anti-HCV) antibody. Participants with history of related disease or positive hepatitis C antibody due to prior resolved disease can be enrolled only if a confirmatory negative hepatitis C RNA test is obtained at screening or within 3 months prior to first dose of study treatment. Other clinically active liver disease of infectious origin.
[0380] 26. Any condition for which, in the opinion of the investigator, participation would not be in the best interest of the participant (eg, compromise the well-being) or that could prevent, limit, or confound the protocol-specified assessments.
[0381] 27. Criteria for Part 2, Cohort A and CohortB only: NSCLC characterized by activating mutations in either the EGFR gene or ALK fusion mutations. Confirmation of the absence of these actionable mutations (by local testing) is required.
[0382] Additional Exclusion Criteria for Parts 1 and 2
[0383] History of Grade 3 or higher toxic effects during prior treatment with immunotherapy or requirement of anti-TNF or anti-IL-6 agents to manage AEs from prior treatment with immunotherapy.
[0384] History of allergy to protein-based therapies or history of any significant drug allergy (such as anaphylaxis, hepatotoxicity, or immune-mediated thrombocytopenia or anemia).
OBJECTIVES AND ENDPOINTS
[0385] The primary objective of Part 1 (dose escalation) is to determine a safe, tolerable, and feasible dose of intratumorally delivered JP-OV-2 and the recommended dose(s) and regimen(s) in combination with cetrelimab in participants with advanced solid tumors. The incidence and severity of AEs, including DLTs, are used to determine the dose(s) and regimen(s) of JP-OV-2 as a monotherapy, and in combination with cetrelimab, to evaluate in Part 2. The primary objective of Part 2 (dose expansion) is to further characterize the safety and tolerability of JP-OV-2 at the selected dose(s) in combination with a PD-1 antibody in select tumor populations. The first 2 cohorts in Part 2 will evaluate the safety of JP-OV-2 at the selected dose(s) in combination with cetrelimab in participants with previously treated metastatic NSCLC with no PD-L1 expression (PD-L1<1%) at the time of original diagnosis (Cohort A), and in treatment-naive participants with metastatic NSCLC with high PD-L1 expression (PD-L1≥50%, Cohort B). Secondary objectives in both parts of the study include determination of the biodistribution of JP-OV-2, viral shedding, immunogenicity, cetrelimab PK, and preliminary clinical activity of JP-OV-2 in combination with cetrelimab.
[0386] Dose Escalation (Part 1): Recommended dose(s) of JP-OV-2 alone and in combination with cetrelimab may be identified such that the isotonic estimate of the DLT rate for monotherapy and the combination is less than or equal to the target rate of 33%.
[0387] Dose Expansion (Part 2): JP-OV-2 in combination with cetrelimab is safe at the recommended dose(s) and regimen(s) in the respective populations.
[0388] Study Outcome Measures:
[0389] Primary Outcome Measures:
[0390] Part 1 : Number of Participants with Dose-Limiting Toxicity (DLT) [ Time Frame: Up to 5 years ]. The DLTs are specific adverse events and are defined as any of the following: non- hematological toxicity and hematologic toxicity.
[0391] Number of Participants with Adverse Events (AEs) by Seventy [ Time Frame: From first dose up to 100 days after last dose of study treatment (up to 5 years) ]. An adverse event is any untoward medical occurrence in a clinical study participant administered a pharmaceutical (investigational or non-investigational) product. An adverse event does not necessarily have a causal relationship with the treatment. Severity will be graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 5.0. Seventy scale ranges from Grade 1: mild, Grade 2: moderate, Grade 3: severe. Grade 4: life-threatening, and Grade 5: death related to adverse event.
[0392] Secondary Outcome Measures:
[0393] Parts 1 and 2: Percentage of Participants With Objective Response (OR) [ Time Frame: Up to 5 years ]. OR is defined as the percentage of participants who have best response of Complete Response (CR) or Partial Response (PR) according to response evaluation criteria in solid tumors (RECIST) v1.1.
[0394] Parts 1 and 2: Percentage of Participants With Disease Control (DC) [ Time Frame: Up to 5 years ]. DC is defined as the percentage of participants who have achieved complete response, partial response, and stable disease according to RECIST v1.1.
[0395] Parts 1 and 2: Duration of Response (DOR) [ Time Frame: Up to 5 years ]. DOR will be calculated among responders from the date of initial documentation of a response to the date of first documented evidence of relapse according to RECIST v1.1, or death due to any cause, whichever occurs first.
[0396] Part 2: Progression Free Survival (PFS) [ Time Frame: From treatment initiation until disease progression or worsening or death due to any cause (up to 5 years) ]. PFS is defined as the time from treatment initiation until disease progression or worsening or death due to any cause.
[0397] Part 2: Overall Sunrival (OS) [ Time Frame: From treatment initiation until death due to any cause (up to 5 years) ] OS is defined as the time from treatment initiation until death due to any cause.
[0398] Parts 1 and 2: Number of JP-OV-2 Genome Copies per Milliliter [ Time Frame: Up to 5 years ]. Viral genome copies of JP-OV-2 collected from samples (that is, blood, urine, oral mucosa, injection sites, and dressings) will be determined by quantitative polymerase chain reaction (qPCR) assays.
[0399] Parts 1 and 2: Payload Concentrations of JP-OV-2 [ Time Frame: Up to 2 years ]. Blood samples will be collected to characterize JP-OV-2 payload concentrations in blood and tumor and will be analyzed using immunoassay.
[0400] Parts 1 and 2: Number of Participants with JP-OV-2 Antibodies [ Time Frame: Up to 2 years ]. Antibodies against JP-OV-2 encoded payloads and against herpes simplex virus type-1 (HSV-1) will be analyzed.
Radiographic Image Assessment
[0401] Baseline disease burden is assessed using CT scans of the chest, abdomen, and pelvis, plus other areas of known disease involvement as appropriate, with IV contrast. Participants who are intolerant of IV contrast agents may have CT scans performed with oral contrast and the reason for not using IV contrast are documented in source documents. Subsequent efficacy evaluations during the study will include radiographic imaging of all disease sites documented at baseline.
[0402] Magnetic resonance imaging may be used to evaluate sites of disease that cannot be adequately imaged using CT scan. In any case where an MRI is desirable it must be the imaging technique used to assess disease at baseline and at all subsequent response evaluations. For all other sites of disease, MRI assessments do not replace the required chest, abdomen, and pelvic CT scans, unless CT scan is contraindicated. Brain MRI is required only if clinically indicated. CT scan of the head can be used if MRI is contraindicated.
Cetrelimab PK
[0403] Serum samples are collected to assess the pharmacokinetics and ADA of cetrelimab in this participant population.
Biodistribution, and Shedding of JP-OV-2
[0404] Whole blood samples are collected to characterize biodistribution of JP-OV-2 and systemic concentrations of payloads. Urine, swabs of the oral mucosa, injected superficial tumors, and exterior dressings are tested for virus shedding via qPCR.
Immunogenicity Assessments
[0405] Immunogenicity assessments are conducted to determine the presence of antibodies against viral antigens expressed by JP-OV-2, the 4 encoded payloads, and cetrelimab. Other analyses may be performed to further characterize the immunogenicity of JP-OV-2, its payloads, and cetrelimab. Samples collected for immunogenicity analyses may additionally be used to evaluate safety or efficacy aspects that address concerns arising during or after the study period. Genetic analyses will not be performed on these serum samples.
Analysis Methods
[0406] The ORR is tabulated with its 95% exact confidence interval. In addition, the number and percentage of participants in each response category are tabulated. For the calculation of ORR, the treated participants who are not evaluable for response are listed as such and considered as non-responders. Both the overall ORR as well as the separate ORRs of injected and uninjected lesions are calculated. [0407] For DOR, PFS, and OS the Kaplan-Meier method is used for descriptive summaries (eg, median and Kaplan-Meier curve).
[0408] The maximum reduction in SOD from baseline is summarized.
Safety Analyses
[0409] The safety of JP-OV-2 is assessed by physical examinations, ECOG performance status, vital signs, clinical safety laboratory tests, viral shedding swabs, pregnancy testing, and monitoring for AEs, including DLTs.
EXAMPLE 3: RESULTS
[0410] The dose escalation study evaluated the treatment of three escalating dose levels of JP- OV-2 administered intratumorally as monotherapy for the initial dose, followed by administration in combination with systemic anti-PD1 therapy (cetrelimab) for subsequent doses. This study involved patients with advanced solid tumors who had experienced relapse after all approved therapies.
[0411] The swim lane plot (FIG. 4) illustrates the duration of treatment (represented by the length of the horizontal bars) and the best clinical response (shown as white circles) for individual patients treated at each dose level. The assessment of response was conducted according to oncology standards using RECIST v1.1, which defines a response as a reduction in total measurable tumor burden by at least 30%. Clinical benefit was defined as stable disease according to RECIST for a minimum of 12 weeks. The repeated intratumoral doses of JP-OV-2, administered every two weeks, were well tolerated. Notably, two patients (100021, 100027) receiving the highest dose achieved a clinical response. Other tumor types showing benefit included colon cancer (Patient 100004 - 12 weeks), thymic cancer (Patient 100008, 28 weeks), pancreatic cancer (Patient 100009, 12 weeks), and anal cancer (Patient 100022, 16 weeks).
[0412] FIG. 5A presents a waterfall plot that shows the post-treatment change in total measurable tumor burden per standard oncology guidelines (RECIST v1.1), with vertical bars representing individual patients. This figure demonstrates a dose-dependent effect of JP-OV-2 on tumor growth control, with effects increasing with ascending doses from left to right; the 10° dose is shown in the first third, the 10'' dose in the second third, and the 108 dose in the final third of FIG. 5 A. At the top dose level of 108, two patients with advanced melanoma achieved a ≥30% reduction in tumor burden, qualifying as a confirmed partial response. Additionally, tumor shrinkage was observed in uninjected lesions (depicted as light bars) as well as in injected lesions (FIG. 5B).
[0413] To assess the expression of JP-OV-2-encoded immune payloads in injected tumors, tumor tissue samples were collected from patients 2 days post-injection of JP-OV-2. FIG. 6A sho ws the quantity of each of the payload proteins detected in these tumor tissues. Pay load proteins were identified in all six tested tissue samples, with all four payloads detectable in four out of six samples and three payloads detected in the remaining two samples. Importantly, the relative levels of payload proteins detected were similar to those observed in preclinical mouse models (FIG. 6B). A direct correlation was found between the amount of payload protein and serum levels of interferon-y (Fig. 3C), indicating immune activation resulting from the effects of the immune payloads.
[0414] FIG. 7 provides data for a responding patient at the top dose level (patient 100027). This patient had metastatic melanoma that was unresponsive to three prior immunotherapy treatments, including anti-PD1 and anti-CTLA-4 therapies. While melanoma typically responds to immunotherapy, patients with refractory disease typically have poor outcomes, with no effective therapies available, and palliative treatments usually lead to disease progression within 2-3 months. Such tumors are considered immunologically “cold” and are not expected to respond to available immunotherapeutic options. Following treatment with JP-OV-2, this patient exhibited a response in both injected and uninjected lesions and has maintained this response for over four months as of the latest data review. The figure includes CT scan images of a representative injected and uninjected lesion (FIG. 7A), quantification of serum levels of JP-OV-2-encoded immune payloads (FIG. 7B), and the corresponding effect on immune activation, represented by serum interferon gamma levels (FIG. 7C). With JP-OV-2 treatment, tumor shrinkage was observed in the first scan taken at six weeks post-treatment, affecting both the injected inguinal lesion and an uninjected lung lesion. Peak levels of three out of four JP-OV-2-encoded payloads were recorded at days 3 and 4, with corresponding peak levels of gamma interferon in serum at day 3, indicative of systemic immune activation.
[0415] FIG. 8 illustrates serum levels of interferon-y at various time points during treatment for patients treated at each JP-OV-2 dose level. Dose-proportional increases in interferon-y levels were observed across the three evaluated doses of JP-OV-2, indicating a dose-dependent effect of JP- OV-2 and its encoded payloads on immune activation in cancer patients. Notably, HSV seronegative patients exhibited higher levels after the first dose, which decreased by the second dose to levels comparable to seropositive patients, correlating with seroconversion.
[0416] T 6 examine the hypothesis that the injection of JP-OV-2 induces both direct and indirect effects mediated by the encoded immune payloads, we evaluated the immune microenvironment in paired tumor tissue samples collected from a lesion before treatment and another sample from the same lesion after three injections of JP-OV-2 (FIG. 9). These samples were obtained from a patient diagnosed with mesothelioma, a disease known for its resistance to immunotherapy and classified as having "cold" immune biology. Consistent with this characterization, the patient previously underwent treatment with anti-PD1 and anti-CTLA-4 therapies but experienced limited benefits before enrolling in this study. The patient was treated with JP-OV-2 at the second dose level, with sequential injections at two different sites when the first site showed response. The patient demonstrated a reduction in total tumor burden, achieving sustained disease control for seven months while receiving a total of twelve injections of JP-OV-2 at two- week intervals, without any skipped doses.
[0417] Immunofluorescence analysis of tumor specimens prior to treatment revealed a cold immune phenotype, characterized by rare CD8 T cells and lowPD-L1 expression. Following three doses of JP-OV-2, the tumor specimen exhibited a more inflamed immune phenotype, marked by significant infiltration of CDS T cells and increased PD-L1 expression, providing direct evidence of the payload's action within the tumor. This finding illustrates the capability of JP-OV-2 to convert a "cold" immunotherapy-resistant tumor into a "hot" immune-responsive tumor (FIG, 9). [0418] In summary, these data indicate that JP-OV-2 treatment is both safe and feasible at the proposed doses. Upon intratumoral administration, JP-OV-2-encoded immune payloads are expressed at the injection sites, enhancing the immune anti-tumor response not only at the injection site but also at uninjected sites when combined with the anti-PD1 agent cetrelimab, resulting in clinical responses in both injected and uninjected tumors.

Claims

What is claimed is:
1. A method of treating an advanced solid tumor in an individual comprising administering to the individual an oncolytic herpes simplex type 1 (HSV-1) virus every two weeks (Q2W) intratumorally, wherein the HSV-1 comprises one or more expression cassettes comprising a polynucleotide encoding hFLT3L protein, a polynucleotide encoding a CTLA-4 binding protein, a polynucleotide encoding a CD40 agonist, and a polynucleotide encoding IL- 12.
2. The method of claim 1, wherein the oncolytic HSV-1 virus comprises: a. a cassette integrated in one or both of the γ34.5 loci comprising in order, from upstream to downstream, a CMV promoter, a polynucleotide encoding hFLT3L, a P2A cleavage sequence, a polynucleotide encoding UL49.5, and a polyadenylation signal; and b. another cassette integrated in the US10-12 locus comprising in order, from upstream to downstream, a polynucleotide comprising a variant US11 gene encoding native US11 protein, an additional polynucleotide encoding native US11 protein, a polynucleotide encoding US 10 protein, a polyadenylation signal that is operably linked to the polynucleotide encoding the US 10 protein, a CMV promoter, a polynucleotide encoding a CTLA-4 binding protein, a polyadenylation signal that is operably linked to the polynucleotide encoding the CTLA-4 binding protein, a polyadenylation signal that is operably linked to a polynucleotide encoding a CD40 agonist, a polynucleotide encoding a CD40 agonist, an AoHV 1 promoter that controls expression of the CD40 agonist, an MMLV promoter that controls expression of an IL- 12, a polynucleotide encoding an IL- 12, and a polyadenylation signal that is operably linked to the polynucleotide encoding the IL- 12, wherein the polynucleotide for hFLT3L encodes the amino acid sequence set forth in SEQ ID NO: 71, the polynucleotide for UL49.5 encodes the amino acid sequence set forth in SEQ ID NO: 82, the polynucleotide for IL-12 encodes the amino acid sequence set forth in SEQ ID NO: 4 , the polynucleotide for CD40 agonist encodes the amino acid sequence set forth in SEQ ID NO: 25, and the polynucleotide for CTLA-4 binding protein encodes the amino acid sequence set forth in SEQ ID NO: 50, the polynucleotide for variant US11 gene comprises the polynucleotide sequence set forth in SEQ ID NO: 204, the additional polynucleotide encoding for US11 encodes the amino acid sequence set forth in SEQ ID NO: 80, and the polynucleotide for US 10 encodes the amino acid sequence set forth in SEQ ID NO: 90.
3. The method of claim 1 or claim 2, wherein the oncolytic HSV-1 virus is JP-OV-2.
4. The method of any one of claims 1-3, wherein the HSV-1 virus is administered to the individual as a monotherapy to treat the advanced solid tumor.
5. The method of any one of claims 1-4, wherein the HSV-1 virus is administered to the individual intratumorally in one or more lesions.
6. The method of any one of claims 1-5, further comprising administering to the individual an antibody that binds to PD-1.
7. The method of any of claims 1-6, wherein the individual does not have a central nervous system solid tumor.
8. The method of any one of claims 1-7, wherein the individual has non-small cell lung cancer (NSCLC).
9. The method of any one of claims 1-8, wherein the individual has relapsed or refractory metastatic NSCLC.
10. The method of any one of claims 1-9, wherein the individual has received all available standard therapy and a solid tumor progressed.
11. The method of any one of claims 1-10, wherein the individual has stage IIIB-IV NSCLC.
12. The method of any one of claims 1-11, wherein the individual was previously treated with (a) an anti-PD-1 or an anti-PD-L1 therapy; and (b) a platinum-based chemotherapy, either as combination or sequentially for a metastatic disease and the metastatic disease has progressed on or after therapy.
13. The method of any one of claims 1-12, wherein the individual cannot tolerate or has previously refused the platinum-based chemotherapy.
14. The method of any one of claims 1-13, wherein the individual is unable to receive the platinum-based chemotherapy and has progressed after the anti-PD-1 or the anti-PD-L1 therapy alone.
15. The method of any one of claims 1-14 wherein the individual has a PD-L1 expression of less than 1% and has previously treated disease .
16. The method of any one of claims 1-14 wherein the individual has a PD-L1 expression of greater than 50% and is treatment-naive.
17. The method of any one of claims 1-16, wherein the HSV-1 virus is administered to the individual at a dose of about 105 about, 106, about 107, or about 108 PFU/mL.
18. The method of any one of claims 1-17, wherein the HSV-1 virus is administered to the individual at a dose of about 105 PFU/mL.
19. The method of any one of claims 1-18, wherein the HSV-1 virus is administered to the individual at a dose of about 106 PFU/mL.
20. The method of any one of claims 1-19, wherein the HSV-1 virus is administered to the individual at a dose of about 107 PFU/mL.
21. The method of any one of claims 1-20, wherein the HSV-1 virus is administered to the individual at a dose of about 108 PFU/mL.
22. The method of any one of claims 1-21, wherein the volume of the HSV-1 injected per lesion is determined based on the longest diameter of the lesion.
23. The method of any one of claims 1-22, wherein the HSV-1 virus is administered to the individual in a maximum volume of 10 ml.
24. The method of any one of claims 1-23, wherein the HSV-1 virus is injected to the individual into a cutaneous, a subcutaneous, or a nodal lesion.
25. The method of claim 24, wherein the HSV-1 virus is administered to the cutaneous, the subcutaneous, or the nodal lesion in a superficial approach with injection under direct visualization and palpation.
26. The method of any one of claims 1-25, wherein the HSV-1 virus is injected to the individual into a transthoracically accessible lung lesion, a transabdominally accessible liver lesion, and/or a subcutaneous soft tissue lesion.
27. The method of claim 26, wherein the HSV-1 virus is administered to the transthoracically accessible lung lesion, the transabdominally accessible liver lesion, and/or the subcutaneous soft tissue lesion in a percutaneous administration approach via imaging guidance.
28. The method of any one of claims 1-27, wherein the HSV-1 virus is administered to a lung lesion or a lymph node accessible via a bronchoscope.
29. The method of any one of claims 1 -28, wherein the individual receives a maximum of 8 doses of the HSV-1 virus.
30. The method of any one of claims 6-29, wherein the antibody that specifically binds to PD-1 comprises a heavy chain variable region (VH) comprising a heavy chain complementarity-determining region (CDRH1) comprising the amino acid sequence set forth in SEQ ID NO: 401, a CDRH2 comprising the amino acid sequence set forth in SEQ ID NO: 402, a CDRH3 comprising the amino acid sequence set forth in SEQ ID NO: 403; and a light chain variable region (VL) comprising a light chain complementarity-determining region (CDRL1) comprising the amino acid sequence set forth in SEQ ID NO: 405, a CDRL2 comprising the amino acid sequence set forth in SEQ ID NO: 406, and a CDRL3 comprising the amino acid sequence set forth in SEQ ID NO: 407.
31. The method of claim 30, wherein the antibody that specifically binds to PD-1 comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 408 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 409.
32. The method of claim 31, wherein the antibody that specifically binds to PD-1 comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 400 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 404.
33. The method of any one of claims 6-32, wherein the antibody that binds to PD-1 is cetrelimab.
34. The method of any one of claims 6-33, wherein the antibody that binds to PD-1 is administered to the individual at a dose of about 480 mg.
35. The method of any one of claims 6-34, wherein the antibody that binds to PD-1 is administered to the individual every four weeks (Q4W).
36. The method of any one of claims 6-35 wherein the antibody that binds to PD-1 is administered to the individual every four weeks (Q4W) for at least about 15 weeks.
37. The method of claim 36, wherein the antibody that binds to PD-1 is administered to the individual four weeks (Q4W) for at most about 2 years.
38. The method of any one of claims 6-37, wherein the antibody that binds to PD-1 is administered intravenously.
39. The method of any one of claims 6-38, wherein the antibody that binds to PD-1 is administered to the individual after the HSV-1 virus.
40. The method of any one of claims 6-39, wherein the antibody that binds to PD-1 is administered to the individual starting in week three of treatment, wherein week one of treatment is the first administration of the HSV-1 virus.
41. The method of any one of claims 6-40, wherein the antibody that binds to PD-1 is administered to the individual starting in week one of treatment, wherein week one of treatment is the first administration of the HSV-1 virus.
42. The method of any one of claims 1-41, wherein the size of the solid tumor is reduced.
43. The method of any one of claims 1-42, wherein the individual has more than one solid tumor.
44. The method of claim 43, wherein the HSV-1 is administered to at least one of the solid tumors.
45. The method of claim 44, wherein the size of a solid tumor other than the at least one solid tumor where the HSV-1 is administered decreases in size following treatment.
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