PROSTATE CANCER VACCINES AND USES THEREOF

SEQUENCE LISTING

The contents of the electronic sequence listing (JBI6595USNP1_Sequence Listing.xml; Size: 111,776 bytes; and Date of Creation: Nov. 23, 2022) is herein incorporated by reference in its entirety.

BACKGROUND

Prostate cancer is the most common non-cutaneous malignancy in men and the second leading cause of death in men from cancer in the western world. Prostate cancer results from the uncontrolled growth of abnormal cells in the prostate gland. Once a prostate cancer tumor develops, androgens such as testosterone promote prostate cancer growth. At its early stages, localized prostate cancer is often curable with local therapy including, for example, surgical removal of the prostate gland and radiotherapy. However, when local therapy fails to cure prostate cancer, as it does in up to a third of men, the disease progresses into incurable metastatic disease.

For many years, the established standard of care for men with malignant castration-resistant prostate cancer (mCRPC) was docetaxel chemotherapy. More recently, abiraterone acetate (ZYTIGA®) in combination with prednisone has been approved for treating metastatic castrate resistant prostate cancer. Androgen receptor (AR)-targeted agents, such as enzalutamide (XTANDI®) have also entered the market for treating metastatic castrate resistant prostate cancer. Platinum-based chemotherapy has been tested in a number of clinical studies in molecularly unselected prostate cancer patients with limited results and significant toxicities. However, there remains a subset of patients who either do not respond initially or become refractory (or resistant) to these treatments. No approved therapeutic options are available for such patients.

SUMMARY

Disclosed herein are methods of treating or preventing prostate cancer in a subject, the methods comprising administering to the subject a treatment regimen comprising two or more vaccines comprising a great ape adenovirus serotype 20 (GAd20) virus that, in turn, comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 and one or more vaccines comprising a Modified Vaccinia Ankara (MVA) virus that, in turn, comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3 to thereby treat or prevent the prostate cancer.

Also provided are methods of treating or preventing prostate cancer in a subject, the methods comprising administering to the subject: a vaccine comprising 1×10¹¹viral particles (VP) of a GAd20 virus at week 0 and about week 3, wherein the GAd20 virus comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1; a vaccine comprising 1×10⁸infectious units (IFU) of an MVA virus at about week 9, wherein the MVA virus comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3; a vaccine comprising 1×10¹¹VP of the GAd20 virus at about week 15 and about week 18; a vaccine comprising 1×10⁸IFU of the MVA virus at about week 24; and a vaccine comprising 1×10⁸IFU of the MVA virus at about week 36, about week 48, and about week 60.

Methods of treating or preventing prostate cancer in a subject are disclosed, wherein the methods comprise administering to the subject: a vaccine comprising 1×10¹¹viral particles (VP) of a GAd20 virus at week 0 and about week 3, wherein the GAd20 virus comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1; a vaccine comprising 1×10⁸infectious units (IFU) of an MVA virus at about week 9, wherein the MVA virus comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3; a vaccine comprising 1×10¹¹VP of the GAd20 virus at about week 15; a vaccine comprising 1×10⁸IFU of the MVA virus at about week 24; and a vaccine comprising 1×10⁸IFU of the MVA virus at about week 36, about week 48, and about week 60.

Also provided are methods of treating or preventing prostate cancer in a subject, the methods comprising administering to the subject: a vaccine comprising 1×10¹¹viral particles (VP) of a GAd20 virus at week 0 and about week 3, wherein the GAd20 virus comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1; a vaccine comprising 1×10⁸infectious units (IFU) of an MVA virus at about week 9, wherein the MVA virus comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3; a vaccine comprising 1×10⁸IFU of the MVA virus at about week 15, about week 18, and about week 24; and a vaccine comprising 1×10⁸IFU of the MVA virus at about week 36, about week 48, and about week 60.

Disclosed herein are methods of treating or preventing prostate cancer in a subject, the methods comprising administering to the subject: a vaccine comprising 1×10¹¹viral particles (VP) of a GAd20 virus at week 0 and about week 3, wherein the GAd20 virus comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1; a vaccine comprising 1×10⁸infectious units (IFU) of an MVA virus at about week 9, wherein the MVA virus comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3; a vaccine comprising 1×10⁸IFU of the MVA virus at about week 15 and about week 24; and a vaccine comprising 1×10⁸IFU of the MVA virus at about week 36, about week 48, and about week 60.

Methods of treating or preventing prostate cancer in a subject are disclosed, wherein the methods comprise administering to the subject: 1 mg/kg to 3 mg/kg of an anti-CTLA4 antibody and a vaccine comprising 1×10¹¹viral particles (VP) of a GAd20 virus at week 0 and about week 3, wherein the GAd20 virus comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1; 1 mg/kg to 3 mg/kg of an anti-CTLA4 antibody and a vaccine comprising 1×10⁸infectious units (IFU) of an MVA virus at about week 9, wherein the MVA virus comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3; 1 mg/kg to 3 mg/kg of an anti-CTLA4 antibody and a vaccine comprising 1×10¹¹VP of the GAd20 virus at about week 15 and about week 18; 1 mg/kg to 3 mg/kg of an anti-CTLA4 antibody and a vaccine comprising 1×10⁸IFU of the MVA virus at about week 24; and 1 mg/kg to 3 mg/kg of an anti-CTLA4 antibody and a vaccine comprising 1×10⁸IFU of the MVA virus at about week 36, about week 48, and about week 60.

Methods of treating or preventing prostate cancer in a subject are provided, wherein the methods comprise administering to the subject: 1 mg/kg to 3 mg/kg of an anti-PD1 antibody and a vaccine comprising 1×10¹¹viral particles (VP) of a GAd20 virus at week 0 and about week 3, wherein the GAd20 virus comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1; 1 mg/kg to 3 mg/kg of an anti-PD1 antibody and a vaccine comprising 1×10⁸infectious units (IFU) of an MVA virus at about week 9, wherein the MVA virus comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3; 1 mg/kg to 3 mg/kg of an anti-PD1 antibody and a vaccine comprising 1×10¹¹VP of the GAd20 virus at about week 15 and about week 18; 1 mg/kg to 3 mg/kg of an anti-PD1 antibody and a vaccine comprising 1×10⁸IFU of the MVA virus at about week 24; and 1 mg/kg to 3 mg/kg of an anti-PD1 antibody and a vaccine comprising 1×10⁸IFU of the MVA virus at about week 36, about week 48, and about week 60.

Also provided are methods of treating or preventing prostate cancer in a subject, the methods comprising administering to the subject: 1 mg/kg to 3 mg/kg of an anti-CTLA4 antibody and a vaccine comprising 1×10¹¹viral particles (VP) of a GAd20 virus at week 0 and about week 3, wherein the GAd20 virus comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1; 1 mg/kg to 3 mg/kg of an anti-CTLA4 antibody and a vaccine comprising 1×10⁸infectious units (IFU) of an MVA virus at about week 9, wherein the MVA virus comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3; 1 mg/kg to 3 mg/kg of an anti-CTLA4 antibody and a vaccine comprising 1×10¹¹VP of the GAd20 virus at about week 15; 1 mg/kg to 3 mg/kg of an anti-CTLA4 antibody and a vaccine comprising 1×10⁸IFU of the MVA virus at about week 24; and 1 mg/kg to 3 mg/kg of an anti-CTLA4 antibody and a vaccine comprising 1×10⁸IFU of the MVA virus at about week 36, about week 48, and about week 60.

Disclosed herein are methods of treating or preventing prostate cancer in a subject, the methods comprising administering to the subject: 1 mg/kg to 3 mg/kg of an anti-PD1 antibody and a vaccine comprising 1×10¹¹viral particles (VP) of a GAd20 virus at week 0 and about week 3, wherein the GAd20 virus comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1; 1 mg/kg to 3 mg/kg of an anti-PD1 antibody and a vaccine comprising 1×10⁸infectious units (IFU) of an MVA virus at about week 9, wherein the MVA virus comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3; 1 mg/kg to 3 mg/kg of an anti-PD1 antibody and a vaccine comprising 1×10¹¹VP of the GAd20 virus at about week 15; 1 mg/kg to 3 mg/kg of an anti-PD1 antibody and a vaccine comprising 1 x 10⁸IFU of the MVA virus at about week 24; and 1 mg/kg to 3 mg/kg of an anti-PD1 antibody and a vaccine comprising 1×10⁸IFU of the MVA virus at about week 36, about week 48, and about week 60.

Disclosed herein are methods of treating or preventing prostate cancer in a subject, the methods comprising administering to the subject: 1 mg/kg to 3 mg/kg of an anti-CTLA4 antibody and a vaccine comprising 1×10¹¹viral particles (VP) of a GAd20 virus at week 0 and about week 3, wherein the GAd20 virus comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1; 1 mg/kg to 3 mg/kg of an anti-CTLA4 antibody and a vaccine comprising 1×10⁸infectious units (IFU) of an MVA virus at about week 9, wherein the MVA virus comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3; 1 mg/kg to 3 mg/kg of an anti-CTLA4 antibody and a vaccine comprising 1×10⁸IFU of the MVA virus at about week 15, about week 18, and about week 24; and 1 mg/kg to 3 mg/kg of an anti-CTLA4 antibody and a vaccine comprising 1×10⁸IFU of the MVA virus at about week 36, about week 48, and about week 60.

Provided herein are methods of treating or preventing prostate cancer in a subject, the method comprising administering to the subject: 1 mg/kg to 3 mg/kg of an anti-PD1 antibody and a vaccine comprising 1×10¹¹viral particles (VP) of a GAd20 virus at week 0 and about week 3, wherein the GAd20 virus comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1; 1 mg/kg to 3 mg/kg of an anti-PD1 antibody and a vaccine comprising 1×10⁸infectious units (IFU) of an MVA virus at about week 9, wherein the MVA virus comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3; 1 mg/kg to 3 mg/kg of an anti-PD1 antibody and a vaccine comprising 1×10⁸IFU of the MVA virus at about week 15, about week 18, and about week 24; and 1 mg/kg to 3 mg/kg of an anti-PD1 antibody and a vaccine comprising 1×10⁸IFU of the MVA virus at about week 36, about week 48, and about week 60.

Methods of treating or preventing prostate cancer in a subject are disclosed, wherein the methods comprise administering to the subject: 1 mg/kg to 3 mg/kg of an anti-CTLA4 antibody and a vaccine comprising 1×10¹¹viral particles (VP) of a GAd20 virus at week 0 and about week 3, wherein the GAd20 virus comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1; 1 mg/kg to 3 mg/kg of an anti-CTLA4 antibody and a vaccine comprising 1×10⁸infectious units (IFU) of an MVA virus at about week 9, wherein the MVA virus comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3; 1 mg/kg to 3 mg/kg of an anti-CTLA4 antibody and a vaccine comprising 1×10⁸IFU of the MVA virus at about week 15 and about week 24; and 1 mg/kg to 3 mg/kg of an anti-CTLA4 antibody and a vaccine comprising 1×10⁸IFU of the MVA virus at about week 36, about week 48, and about week 60.

Provided herein are methods of treating or preventing prostate cancer in a subject, the method comprising administering to the subject: 1 mg/kg to 3 mg/kg of an anti-PD1 antibody and a vaccine comprising 1×10¹¹viral particles (VP) of a GAd20 virus at week 0 and about week 3, wherein the GAd20 virus comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1; 1 mg/kg to 3 mg/kg of an anti-PD1 antibody and a vaccine comprising 1×10⁸infectious units (IFU) of an MVA virus at about week 9, wherein the MVA virus comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3; 1 mg/kg to 3 mg/kg of an anti-PD1 antibody and a vaccine comprising 1×10⁸IFU of the MVA virus at about week 15 and about week 24; and 1 mg/kg to 3 mg/kg of an anti-PD1 antibody and a vaccine comprising 1×10⁸IFU of the MVA virus at about week 36, about week 48, and about week 60.

BRIEF DESCRIPTION OF THE DRAWINGS

The summary, as well as the following detailed description, is further understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosed methods, there are shown in the drawings exemplary embodiments of the methods; however, the methods is not limited to the specific embodiments disclosed. In the drawings:

FIG. 1A and FIG. 1B illustrate exemplary dosing schedules.

FIG. 2 illustrates the number of cynomolgus monkeys exhibiting prostate cancer neo-antigen specific IFNγ+ T cells (SFU/10⁶cells) (responders) at week 2 and week 6 after receiving a single dose (“single prime”) or two doses (“double prime”) of the disclosed GAd20 vaccine.

FIG. 3 illustrates the antigen specific T cell response in cynomolgus monkeys after receiving a double prime of the GAd20 vaccine followed by an MVA vaccine boost at the indicated time points.

FIG. 4 illustrates the number of cynomolgus monkeys exhibiting prostate cancer neo-antigen specific IFNy+T cells (SFU/10⁶cells) (responders) at week 4 after receiving a single dose (“single prime”) of GAd20 alone or in combination with an anti-CTLA4 antibody (αCTLA4).

FIG. 5 illustrates the prostate cancer neo-antigen specific IFNγ+ T cell (SFU/10⁶cells) response in cynomolgus monkeys up to week 22 after receiving the indicated dosing schedules.

FIG. 6 illustrates the polyfunctional (IFNγ/TNFα double positive) CD8+1 T cell response in cynomolgus monkeys following the administration of GAd20/GAd20/MVA (at weeks 0, 4, 12) alone (circle) or in combination with an anti-CTLA4 antibody (square).

FIG. 7 illustrates the effector memory neo-antigen specific CD8+ T cell response in cynomolgus monkeys following the administration of GAd20/GAd20/MVA (at weeks 0, 4, 12) alone (circle) or in combination with an anti-CTLA4 antibody (square) at week 21.

FIG. 8 illustrates the breadth of the CD8+ T cell response in cynomolgus monkeys following the administration of GAd20/GAd20/MVA (at weeks 0, 4, 12) alone or in combination with an anti-CTLA4 antibody.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The disclosed methods may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures, which form a part of this disclosure. It is to be understood that the disclosed methods are not limited to the specific methods described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed methods.

Unless specifically stated otherwise, any description as to a possible mechanism or mode of action or reason for improvement is meant to be illustrative only, and the disclosed methods are not to be constrained by the correctness or incorrectness of any such suggested mechanism or mode of action or reason for improvement.

Where a range of numerical values is recited or established herein, the range includes the endpoints thereof and all the individual integers and fractions within the range, and also includes each of the narrower ranges therein formed by all the various possible combinations of those endpoints and internal integers and fractions to form subgroups of the larger group of values within the stated range to the same extent as if each of those narrower ranges was explicitly recited. Where a range of numerical values is stated herein as being greater than a stated value, the range is nevertheless finite and is bounded on its upper end by a value that is operable within the context of the methods as described herein. Where a range of numerical values is stated herein as being less than a stated value, the range is nevertheless bounded on its lower end by a non-zero value. It is not intended that the scope of the methods be limited to the specific values recited when defining a range. All ranges are inclusive and combinable.

When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise.

It is to be appreciated that certain features of the disclosed methods which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed methods that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.

As used herein, the singular forms “a,” “an,” and “the” include the plural.

Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

The term “about” is used to encompass variations of ±10% or less, variations of ±5% or less, variations of ±1% or less, variations of ±0.5% or less, or variations of ±0.1% or less from the specified value

The term “comprising” is intended to include examples encompassed by the terms “consisting essentially of” and “consisting of”; similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.”

“Administered with” means that two or more therapeutics (such as a virus and an antibody) can be administered to a subject together in a mixture, concurrently as single agents, or sequentially as single agents in any order.

“Treat,” “treatment,” and like terms refer to both therapeutic treatment and prophylactic or preventative measures, and includes reducing the severity and/or frequency of symptoms of prostate cancer, eliminating symptoms and/or the underlying cause of the symptoms of prostate cancer, reducing the frequency or likelihood of symptoms of prostate cancer and/or their underlying cause, and improving or remediating damage caused, directly or indirectly, by prostate cancer. Treatment also includes prolonging survival as compared to the expected survival of a subject not receiving treatment. Subjects to be treated include those that have prostate cancer as well as those prone to have prostate cancer or those in which prostate cancer is to be prevented.

Disclosed herein are methods of treating or preventing prostate cancer in a subject, wherein the prostate cancer comprises one or more prostate cancer neoantigens encoding an amino acid sequence of SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, or 93. The disclosed vaccines (e.g., those comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1 or 3) can be used to treat or prevent prostate cancers that express one or more of these neoantigens. The methods of treating or preventing prostate cancer can comprise administering to the subject a treatment regimen comprising:

two or more vaccines comprising a great ape adenovirus serotype 20 (GAd20) virus that, in turn, comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 and

one or more vaccines comprising a Modified Vaccinia Ankara (MVA) virus that, in turn, comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3 to thereby treat or prevent the prostate cancer.

GAd20 is an adenovirus that infects gorilla (Gorilla), and can be isolated from stool samples of the gorilla. The GAd20 can be engineered to comprise at least one functional deletion or a complete removal of a gene product that is essential for viral replication, such as one or more of the adenoviral regions E1, E2 and E4, therefore rendering the adenovirus to be incapable of replication. The deletion of the E1 region may comprise deletion of EIA, EIB 55K or EIB 21K, or any combination thereof. Replication deficient adenoviruses are propagated by providing the proteins encoded by the deleted region(s) in trans by the producer cell by utilizing helper plasmids or engineering the produce cell to express the required proteins. Adenovirus vectors may also have a deletion in the E3 region, which is dispensable for replication, and hence such a deletion does not have to be complemented. The GAd20 of the disclosure may comprise a functional deletion or a complete removal of the E1 region and at least part of the E3 region. The GAd20 may further comprise a functional deletion or a complete removal of the E4 region and/or the E2 region. Suitable producer cells that can be utilized are human retina cells immortalized by E1, e.g. 911 or PER.C6 cells (see, e.g., U.S. Pat. No. 5,994,128), E1-transformed amniocytes (See, e.g., EP 1230354), E1-transformed A549 cells (see e.g. Int. Pat. Publ. No. WO1998/39411, U.S. Pat. No. 5,891,690). The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 may be inserted into a site or region (insertion region) in the viral genome that does not affect virus viability of the resultant recombinant virus. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 may be inserted into the deleted E1 region in parallel (transcribed 5′ to 3′) or anti-parallel (transcribed in a 3′ to 5′ direction relative to the vector backbone) orientation. In addition, appropriate transcriptional regulatory elements that are capable of directing expression of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 in the mammalian host cells that the virus is being prepared for use may be operatively linked to the nucleotide sequence. “Operatively linked” sequences include both expression control sequences that are contiguous with the nucleic acid sequences that they regulate and regulatory sequences that act in trans, or at a distance to control the regulated nucleic acid sequence.

Recombinant GAd20 particles may be prepared and propagated according to any conventional technique in the field of the art (e.g., Int. Pat. Publ. No. WO1996/17070) using a complementation cell line or a helper virus, which supplies in trans the missing viral genes necessary for viral replication. The cell lines 293 (Graham et al., 1977, J. Gen. Virol. 36: 59-72), PER.C6 (see e.g. U.S. Pat. No. 5,994,128), E1 A549 and 911 are commonly used to complement E1 deletions. Other cell lines have been engineered to complement defective vectors (Yeh, et al., 1996, J. Virol. 70: 559-565; Kroughak and Graham, 1995, Human Gene Ther. 6: 1575-1586; Wang, et al., 1995, Gene Ther. 2: 775-783; Lusky, et al., 1998, J. Virol. 72: 2022-203; EP 919627 and Int. Pat. Publ. No. WO1997/04119). The GAd20 particles may be recovered from the culture supernatant but also from the cells after lysis and optionally further purified according to standard techniques (e.g., chromatography, ultracentrifugation, as described in Int. Pat. Publ. No. WO1996/27677, Int. Pat. Publ. No. WO1998/00524, Int. Pat. Publ. No. WO1998/26048 and Int. Pat. Publ. No. WO2000/50573). The construction and methods for propagating adenoviral vectors, such as GAd20, are also described in for example, U.S. Pat. Nos. 5,559,099, 5,837,511, 5,846,782, 5,851,806, 5,994,106, 5,994,128, 5,965,541, 5,981,225, 6,040,174, 6,020,191, and 6,113,913.

MVA originates from the dermal vaccinia strain Ankara (Chorioallantois vaccinia Ankara (CVA) virus) that was maintained in the Vaccination Institute, Ankara, Turkey for many years and used as the basis for vaccination of humans. However, due to the often severe post-vaccinal complications associated with vaccinia viruses (VACV), there were several attempts to generate a more attenuated, safer smallpox vaccine.

MVA has been generated by 516 serial passages on chicken embryo fibroblasts of the CVA virus (see Meyer et al., J. Gen. Virol., 72: 1031-1038 (1991) and U.S. Pat. No. 10,035,832). As a consequence of these long-term passages the resulting MVA virus deleted about 31 kilobases of its genomic sequence and, therefore, was described as highly host cell restricted to avian cells (Meyer, H. et al.,; Meisinger-Henschel et al., J. Gen. Virol. 88, 3249-3259, 2007). Comparison of the MVA genome to its parent, CVA, revealed 6 major deletions of genomic DNA (deletion I, II, III, IV, V, and VI), totaling 31,000 basepairs. (Meyer et al., J. Gen. Virol. 72:1031-8 (1991)). It was shown in a variety of animal models that the resulting MVA was significantly avirulent (Mayr, A. & Danner, K. Vaccination against pox diseases under immunosuppressive conditions, Dev. Biol. Stand. 41: 225-34, 1978). Being that many passages were used to attenuate MVA, there are a number of different strains or isolates, depending on the passage number in CEF cells, such as MVA 476 MG/14/78, MVA-571, MVA-572, MVA-574, MVA-575 and MVA-BN. MVA 476 MG/14/78 is described for example in Int. Pat. Publ. No. WO2019/115816A1. MVA-572 strain was deposited at the European Collection of Animal Cell Cultures (“ECACC”), Health Protection Agency, Microbiology Services, Porton Down, Salisbury SP4 OJG, United Kingdom (“UK”), under the deposit number ECACC 94012707 on Jan. 27, 1994. MVA-575 strain was deposited at the ECACC under deposit number ECACC 00120707 on Dec. 7, 2000; MVA-Bavarian Nordic (“MVA-BN”) strain was deposited at the ECACC under deposit number V00080038 on Aug. 30, 2000. The genome sequences of MVA-BN and MVA-572 are available at GenBank (Accession numbers DQ983238 and DQ983237, respectively). The genome sequences of other MVA strains can be obtained using standard sequencing methods.

The MVA can be derived from any MVA strain or further derivatives of the MVA strain. A further exemplary MVA strain is deposit VR-1508, deposited at the American Type Culture collection (ATCC), Manassas, Va. 20108, USA. “Derivatives” of MVA refer to viruses exhibiting essentially the same characteristics as the parent MVA, but exhibiting differences in one or more parts of their genomes. In some embodiments, the MVA vector is derived from MVA 476 MG/14/78 . In some embodiments, the MVA vector is derived from MVA-571. In some embodiments, the MVA vector is derived from MVA-572. In some embodiments, the MVA vector is derived from MVA-574. In some embodiments, the MVA vector is derived from MVA-575. In some embodiments, the MVA vector is derived from MVA-BN.

The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3 may be inserted into a site or region (insertion region) in the MVA viral genome that does not affect virus viability of the resultant recombinant virus. Such regions can be readily identified by testing segments of virus DNA for regions that allow recombinant formation without seriously affecting virus viability of the recombinant virus. The thymidine kinase (TK) gene is an insertion region that may be used and is present in many viruses, such as in all examined poxvirus genomes. Additionally, MVA contains 6 natural deletion sites, each of which may be used as insertion sites (e.g. deletion I, II, III, IV, V, and VI; see e.g. U.S. Pat. Nos. 5,185,146 and 6.440,442). One or more intergenic regions (IGR) of the MVA may also be used as an insertion site, such as IGRs IGR07/08, IGR 44/45, IGR 64/65, IGR 88/89, IGR 136/137, and IGR 148/149 (see e.g. U.S. Pat. Publ. No. 2018/0064803). Additional suitable insertion sites are described in Int. Pat. Publ. No. WO2005/048957.

MVA virus can be prepared as previously described (Piccini, et al., 1987, Methods of Enzymology 153: 545-563; U.S. Pat. NoS. 4,769,330; 4,772,848; 4,603,112; 5,100,587 and 5,179,993). In an exemplary method, the DNA sequence to be inserted into the viral genome can be placed into an E. coli plasmid construct into which DNA homologous to a section of DNA of the MVA has been inserted. Separately, the DNA sequence to be inserted can be ligated to a promoter. The promoter-gene linkage can be positioned in the plasmid construct so that the promoter-gene linkage is flanked on both ends by DNA homologous to a DNA sequence flanking a region of MVA DNA containing a non-essential locus. The resulting plasmid construct can be amplified by propagation within E. coli bacteria and isolated. The isolated plasmid containing the DNA gene sequence to be inserted can be transfected into a cell culture, e.g., of chicken embryo fibroblasts (CEFs), at the same time the culture is infected with MVA. Recombination between homologous MVA DNA in the plasmid and the viral genome, respectively, can generate an MVA modified by the presence of foreign DNA sequences. MVA particles may be recovered from the culture supernatant or from the cultured cells after a lysis step (e.g., chemical lysis, freezing/thawing, osmotic shock, sonication and the like). Consecutive rounds of plaque purification can be used to remove contaminating wild type virus. Viral particles can then be purified using the techniques known in the art (e.g., chromatographic methods or ultracentrifugation on cesium chloride or sucrose gradients).

The methods can further comprise administering one or more vaccines comprising the GAd20 virus, one or more vaccines comprising the MVA virus, or one or more vaccines comprising the GAd20 virus and one or more vaccines comprising the MVA virus. In some embodiments, the methods can further comprise administering the treatment regimen two or more times. After the initial treatment regimen, for example, the methods can comprise administering two vaccines comprising the GAd20 virus and one vaccine comprising the MVA virus. In some embodiments, the methods can further comprise administering one vaccine comprising the GAd20 virus and one vaccine comprising the MVA virus. In some embodiments, the methods can further comprise administering one or more vaccines comprising the MVA virus. The methods can further comprise, for example, administering three vaccines comprising the MVA virus. The methods can further comprise, for example, administering two vaccines comprising the MVA virus.

Suitable amounts of the GAd20 virus can comprise about 1×10⁹viral particles (VP) to about 1×10¹³VP of the GAd20 virus.

Suitable amounts of the MVA virus can comprise about 1×10⁶infectious units (IFU) to about 1×10¹⁰IFU of the MVA virus.

The methods can further comprise administering an anti-CTLA4 antibody. The anti-CTLA4 antibody can be administered with the vaccines comprising the GAd20 virus, with the vaccines comprising the MVA virus, or both. Suitable amounts of the anti-CTLA4 antibody comprise about 0.5 mg/kg to about 5 mg/kg. Any antagonistic anti-CTLA4 antibody can be used in the disclosed methods. Suitable anti-CTLA4 antibodies for use in the disclosed methods include, without limitation, human anti-CTLA4 antibodies, mouse anti-CTLA4 antibodies, mammalian anti-CTLA4 antibodies, humanized anti-CTLA4 antibodies, monoclonal anti-CTLA4 antibodies, polyclonal anti-CTLA4 antibodies, and chimeric anti-CTLA4 antibodies. Non-limiting examples of anti-CTLA4 antibodies include Ipilimumab and tremelimumab.

The methods can further comprise administering an anti-PD-1 antibody. The anti-PD-1 antibody can be administered with the vaccines comprising the GAd20 virus, with the vaccines comprising the MVA virus, or both. Suitable amounts of the anti-PD-1 antibody comprise about 0.5 mg/kg to about 5 mg/kg. Any antagonistic anti-PD-1 antibody can be used in the disclosed methods. Suitable anti-PD-1 antibodies for use in the disclosed methods include, without limitation, human anti-PD-1 antibodies, mouse anti-PD-1 antibodies, mammalian anti-PD-1 antibodies, humanized anti-PD-1 antibodies, monoclonal anti-PD-1 antibodies, polyclonal anti-PD-1 antibodies, and chimeric anti-PD-1 antibodies. Non-limiting examples of anti-PD-1 antibodies include cetrelimab, pembrolizumab, nivolumab, sintilimab, cemiplimab, toripalimab, camrelizumab, tislelizumab, dostralimab, spartalizumab, prolgolimab, balstilimab, budigalimab, sasanlimab, avelumab, atezolizumab, durvalumab, envafolimab, and iodapolimab.

The methods can comprise administering a vaccine comprising the GAd20 virus at week 0 and about week 3 and administering a vaccine comprising the MVA virus at about week 9, and:

- Administering a vaccine comprising the GAd20 virus at about week 15 and about week 18 and administering a vaccine comprising the MVA virus at about week 24;
- Administering a vaccine comprising the GAd20 virus at about week 15 and administering a vaccine comprising the MVA virus at about week 24;
- Administering a vaccine comprising the MVA virus at about week 15, about week 18, and about week 24; or
- Administering a vaccine comprising the MVA virus at about week 15 and about week 24.

In some embodiments, the methods further comprise administering a vaccine comprising the MVA virus at about week 36, about week 48, and about week 60. The methods can further comprise administering one or more further vaccines comprising the MVA virus.

The methods can comprise administering a vaccine comprising 1×10¹¹VP of the GAd20 virus at week 0 and about week 3 and administering a vaccine comprising 1×10⁸IFU of the MVA virus at about week 9, and:

- Administering a vaccine comprising 1×10¹¹VP of the GAd20 virus at about week 15 and about week 18 and administering a vaccine comprising 1×10⁸IFU of the MVA virus at about week 24;
- Administering a vaccine comprising 1×10¹¹VP of the GAd20 virus at about week 15 and administering a vaccine comprising 1×10⁸IFU of the MVA virus at about week 24;
- Administering a vaccine comprising 1×10⁸IFU of the MVA virus at about week 15, about week 18, and about week 24; or
- Administering a vaccine comprising 1×10⁸IFU of the MVA virus at about week 15 and about week 24.

In some embodiments, the methods further comprise administering a vaccine comprising 1×10⁸IFU of the MVA virus at about week 36, about week 48, and about week 60. The methods can further comprise administering one or more further vaccines comprising 1×10⁸IFU of the MVA virus.

The disclosed methods can comprise the exemplary treatment schedules provided in Table 1 below:

TABLE 1

Exemplary treatment schedules

Cycle 1*
Cycle 2
Maintenance

Dose 1
Dose 2
Dose 3
Dose 1
Dose 2
Dose 3
Dose 1
Dose 2
Dose 3

(wk 0)
(wk 3)
(wk 9)
(wk 15)
(wk 18)
(wk 24)
(wk 36)
(wk 48)
(wk 60)
Dose 4
Dose 5

1
GAd20
GAd20
MVA
GAd20
GAd20
MVA
MVA
MVA
MVA
MVA
MVA

2
GAd20
GAd20
MVA
GAd20

MVA
MVA
MVA
MVA
MVA
MVA

3
GAd20
GAd20
MVA
MVA
MVA
MVA
MVA
MVA
MVA
MVA
MVA

4
GAd20
GAd20
MVA
MVA

MVA
MVA
MVA
MVA
MVA
MVA

*Cycle 1 is referred to herein as a “treatment regimen.”