WO2015092710A1 - Contralateral co-administration of vaccines - Google Patents
Contralateral co-administration of vaccines Download PDFInfo
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- WO2015092710A1 WO2015092710A1 PCT/IB2014/067022 IB2014067022W WO2015092710A1 WO 2015092710 A1 WO2015092710 A1 WO 2015092710A1 IB 2014067022 W IB2014067022 W IB 2014067022W WO 2015092710 A1 WO2015092710 A1 WO 2015092710A1
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- A—HUMAN NECESSITIES
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- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
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- A—HUMAN NECESSITIES
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- A61K39/0005—Vertebrate antigens
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- A—HUMAN NECESSITIES
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- A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
Definitions
- the present invention relates to a method of raising an immune response against a polypeptide antigen, comprising the co-administration of an immunogenic polypeptide and a polynucleotide encoding a polypeptide immunogen, where the agents are administered concomitantly (co-administered) and at different anatomic sites of an individual subject in need of treatment.
- the methods are capable of inducing CD4+ and CD8+ T-cell responses as well as antibody responses in subjects, without recourse to complex sequential prime-boost schedules.
- Methods of vaccinating to raise an immune response include the administration of a protein or polypeptide which stimulates an aspect of the immune response in vivo, as well as the administration of a polynucleotide which, once properly administered to a subject, is transcribed and translated into a protein or polypeptide which stimulates an aspect of the immune response in vivo.
- Prime-boost vaccination is a strategy for eliciting an immune response to a target antigen (the immunogen), which can use multiple methods of administering the antigen.
- the immunogen a target antigen
- One administration of the antigen is considered to 'prime' the immune system; the administration of the same antigen (by a different method) is considered to 'boost' the immune response.
- Prime-boost vaccination has been shown to be capable of inducing high levels of antigen-specific CD4 + and CD8 + cells.
- the 'priming' administration and the 'boosting' administration occur sequentially in time. The administration may occur at the same anatomic site (e.g., the same limb) or at separate anatomic sites (e.g., contralateral ⁇ , using left and right limbs; or ipsilaterally using sites on different limbs).
- Heterologous prime-boost protocols may, for example, comprise administration of a
- composition comprising a polypeptide immunogen
- administration of a pharmaceutical composition comprising a polynucleotide that expresses a polypeptide immunogen.
- polynucleotide encoding a second immunogenic polypeptide derived from the same pathogen; and (c) an adjuvant.
- the mammalian immune response has two key components: the humoral response and the cell-mediated response.
- the humoral response involves the generation of circulating antibodies which bind to the antigen to which they are specific, thereby neutralising the antigen and initiating its clearance by cells of the immune system.
- the cell-mediated response involves the interplay of numerous different types of cells, including T cells. T cells are divided into subsets, including the CD4+ and CD8+ T cells.
- prime-boost vaccine strategies require multiple vaccinations and can be burdensome or unviable, especially in mass immunization programs or programs in the developing world.
- An aspect of the present invention is a method of raising an immune response in a subject, by administering (a) a polypeptide immunogen and (b) a polynucleotide vector encoding the same polypeptide immunogen, where the polypeptide immunogen and the polynucleotide-based vector are administered at about the same time, in different limbs of the subject.
- a further aspect of the present invention is a method of treating cancer in a subject in need of such treatment, by administering (a) a polypeptide immunogen selected from a tumor- associated antigen, an immunogenic fragment of a tumor-associated antigen, and a fusion protein comprising a tumor-associated antigen or an immunogenic fragment of a tumor- associated antigen, and (b) a polynucleotide vector encoding the same polypeptide immunogen, where the polypeptide immunogen and the polynucleotide-based vector are administered at about the same time, in different limbs of the subject, and where the subject's cancer expresses the tumor-associated antigen.
- a further aspect of the present invention is a method of increasing an immune response in a subject against a polypeptide immunogen, by administering (a) a polypeptide immunogen and (b) a polynucleotide vector encoding the same polypeptide immunogen, where the polypeptide immunogen and the polynucleotide-based vector are administered at about the same time, in different limbs of the subject, and where the immune response is increased compared to that which would be achieved by administration of the polypeptide immunogen and the polynucleotide vector at about the same time to the same limb of the subject.
- a further aspect of the present invention is a composition
- a composition comprising a polynucleotide vector encoding a polypeptide immunogen, for use in raising an immune response in a subject by coadministration with the same polypeptide immunogen, where the polynucleotide vector and the polypeptide immunogen are administered in different limbs of the subject at about the same time.
- a further aspect of the present invention is a composition comprising a polypeptide immunogen, for use in raising an immune response in a subject by coadministration with a composition comprising a polynucleotide vector encoding the same polypeptide immunogen, where the polynucleotide vector and the polypeptide immunogen are administered in different limbs of the subject at about the same time.
- a further aspect of the present invention is a use of a composition comprising a polynucleotide vector encoding a polypeptide immunogen, in the manufacture of a medicament for use in raising an immune response in a subject by coadministration with a composition comprising the same polypeptide immunogen, where the polynucleotide vector and the polypeptide immunogen are administered in different limbs of the subject at about the same time.
- a further aspect of the present invention is a use of a composition comprising a polypeptide immunogen, in the manufacture of a medicament for use in raising an immune response in a subject by coadministration with a composition comprising a polynucleotide vector encoding the same polypeptide immunogen, where the polynucleotide vector and the polypeptide immunogen are administered in different limbs of the subject at about the same time.
- a further aspect of the present invention is a kit comprising a polynucleotide vector encoding an immunogenic polypeptide, for use in raising an immune response in a subject when coadministered with a pharmaceutical composition comprising the same immunogenic polypeptide, where the coadministration occurs in different limbs of the subject.
- a further aspect of the present invention is a kit comprising (a) a polynucleotide vector encoding an immunogenic polypeptide and (b) a composition comprising the same
- immunogenic polypeptide for use in raising an immune response in a subject when the polynucleotide vector and the immunogenic polypeptide are coadministered to different limbs of the subject.
- Figure 1 graphs the MAGEA3 specific CD8 T cell responses at week two and week four, post prime, analyzed by pentamer staining on blood samples. Each bar represents the mean (with SEM) of blood tested from each mouse in the group. The Y axis is the percentage of CD8 cells that were MAGEA3 specific.
- the six columns shown for week 2 correspond, from left to right, with groups 1-6 as shown in Table 1.
- the six columns shown for week 4 also correspond, from left to right, with groups 1-6 as shown in Table 1.
- Figure 2 graphs the MAGEA3 specific CD4 and CD8 T cell responses at week four, post prime, where analysis was conducted by ICS on splenocytes. Each mouse in groups 1-4 (as set forth on Table 1) was tested. The Y-axis is percentage of IFN gamma (IFNy) positive CD4 cells (circles) or CD8 cells (squares).
- IFNy IFN gamma
- Figure 3 graphs the MAGEA3 specific D8 responses (by pentamer staining of blood samples) at week two, week four, week five, week eight, week nine and week ten post prime. Induction of a strong response after the second boost is demonstrated in each tested regimen. Data was generated with pooled blood samples from each group, except in week5 and week 9 when individual samples were tested.
- Figure 4 graphs MAGEA3 specific CD8 responses as detected by ICS analysis of splenocytes, at week ten (two weeks after the second boost). The data demonstrates that regimens based on contralateral co-administration of viral vectors encoding a tumor-associated antigen and a tumor-associated antigen polypeptide induced strong CD4 and CD8 responses.
- the present invention is based on the finding that concomitant administration of a polypeptide-based immunogen and a polynucleotide-based immunogen at separate anatomic sites (e.g., in contralateral limbs) results in an increase in the CD4+ and/or CD8+ response obtained, compared to the concomitant administration of such immunogens at the same anatomic site (immune response determined, for example, using the assay techniques disclosed herein). Accordingly, concomitant administration of a polypeptide-based immunogen and a polynucleotide-based immunogen in contralateral limbs of a subject is an aspect of the present invention.
- Polynucleotide-based immunogens used in the present invention include those that encode a polypeptide consisting of an amino acid sequence identical to that of the administered polypeptide immunogen, those that encode a polypeptide comprising an amino acid sequence identical to that of the administered polypeptide immunogen, those that encode an immunogenic fragment of the administered polypeptide immunogen, or those that encode a fusion protein comprising a sequence identical to that of the administered polypeptide immunogen or of an immunogenic fragment thereof.
- Administration of the polynucleotide-based immunogen results in an immune response to the encoded polypeptide.
- the polypeptide encoded by the polynucleotide- based immunogen is identical to the administered polypeptide based immunogen, i.e., has 100% sequence identity.
- the polypeptide encoded by the polynucleotide-based immunogen is substantially the same as the administered polypeptide- based immunogen, i.e., the polypeptide encoded by the administered polynucleotide and the polypeptide-based immunogen have an overall sequence identity of at least 90%, e.g. 95% or more, e.g. 98% or more, or e.g. 99% or more over the length of the immunogenic polypeptides.
- reference to administration of "the same" polypeptide as both a polypeptide- based immunogen and as a polynucleotide-based immunogen refers to administration of both the polypeptide, and a polynucleotide that encodes a polypeptide having the same amino acid sequence.
- the polypeptide-based immunogen is a tumor associated antigen (TA antigen), and is administered for the treatment or prophylaxis of a cancer that expresses the TA antigen.
- TA antigen tumor associated antigen
- MAGE- A3 protein or polypeptide, or immunogenic fragment thereof, or a fusion protein comprising a MAGE- A3 protein or polypeptide, or immunogenic fragment thereof is administered for the treatment or prophylaxis of a MAGE-A3 expressing tumor.
- Such tumors may include, e.g., melanoma, non-small cell lung cancer, liver (hepatic) cancer, and bladder cancer
- a human subject is treated by the concomitant administration of a MAGE- A3 polypeptide immunogen and a polynucleotide vector , such as a viral vector, that encodes a MAGE-A3 polypeptide immunogen, for the treatment or prophylaxis of a MAGE-A3 expressing tumor.
- a polynucleotide vector such as a viral vector
- raising an immune response refers to the production in a subject of CD4+ cells, CD8+ cells, and/or antibodies specific to the administered polypeptide immunogen.
- the methods of the present invention better stimulate production of CD4+ and/or CD8+ cells and /or antibodies relative to other vaccination protocols/schedules using the same polypeptide immunogens. By 'better stimulates' is meant that the intensity and/or persistence and/or breadth of the immune response is enhanced.
- cancer refers to malignant neoplasms, including those arising from epithelial tissues and those arising from mesodermal tissues.
- a "tumor” is a mass of cancerous tissue. Anatomic site of immunization / timing of administration
- Methods of immunization according to the present invention comprise the concomitant administration (co-administration) to a subject of an antigenic protein/polypeptide (a)
- polypeptide-based immunogenic formulation in combination with the administration of a nucleic acid encoding an antigenic protein/polypeptide (a polynucleotide-based immunogenic formulation), where the formulations are administered at separate anatomic sites.
- compositions are administered to the same subject at the same time
- “At about the same time” encompasses sequential administration where the period between administrations is due only to the speed of the individual administering the active agents, rather than an intentional period of delay between administrations, e.g., the time period necessary for a single health care practitioner to administer a first composition according to accepted clinical practices and standards, and then administer a second composition according to accepted clinical practices and standards.
- “at about the same time” encompasses administrations within a time period of fifteen minutes or less, thirty minutes or less, one hour or less, two hours or less, six hours or less, up to about twelve hours or less.
- concomitant administration occurs in a time period of no more than about thirty minutes, or no more than about one hour, or no more than about two hours, and does not extend beyond 12 hours.
- the administered immunogenic compositions used in the present invention may include an adjuvant, as is known in the art.
- the health care provider may mix the adjuvant and immunogen immediately prior to administration to the subject.
- the polypeptide-based immunogenic composition is administered to one limb of the subject, and the polynucleotide-based immunogenic
- composition is administered to a contralateral limb of the subject (e.g., right arm and left arm, or left leg and right arm).
- a contralateral limb of the subject e.g., right arm and left arm, or left leg and right arm.
- polypeptide-based immunogenic composition is administered to one limb of the subject, and the polynucleotide-based
- immunogenic composition is administered to a separate limb of the subject, including ipsilateral limbs (e.g., right arm and right leg, left arm and left leg).
- ipsilateral limbs e.g., right arm and right leg, left arm and left leg.
- administration to 'the same anatomic site' refers to administration in the same limb, or within an anatomic area that drains to the same set of lymph nodes.
- Administration may be by any suitable route as is known in the art, and includes intramuscular injection, subcutaneous injection, intradermal delivery, epidermal delivery and transdermal delivery.
- a method of raising an immune response which comprises administering (i) one or more immunogenic polypeptides, and (ii) one or more polynucleotide-based vectors (e.g., viral vectors) comprising one or more heterologous polynucleotides encoding one or more immunogenic polypeptides; wherein (i) and (ii) are administered concomitantly but at separate anatomic sites.
- polynucleotide-based vectors e.g., viral vectors
- heterologous polynucleotides encoding one or more immunogenic polypeptides
- polynucleotide vector may consist of or comprise the same, or substantially the same, amino acid sequence as the administered immunogenic polypeptide, or consist of or comprise an immunogenic fragment of the administered polypeptide immunogen.
- polypeptide-based immunogen is contained in a pharmaceutical comnposition comprising additional pharmaceutically acceptable components, e.g. excipients and carriers.
- polynucleotide-based immunogen is contained in a pharmaceutical comnposition comprising additional pharmaceutically acceptable components, e.g., excipients and carriers.
- Methods according to the invention may involve use of more than one immunogenic polypeptide and/or more than one polynucleotide-based immunogen.
- the polypeptide-based immunogen is administered in such an amount as is capable of inducing an immune response.
- the immune response may be useful as as prophylactic (to prevent disease, including preventing a recurrence of disease) or as a therapeutic (to treat existing disease).
- a desired immune response is achieved by a single concomitant administration (co-administration) of polypeptide-based and polynucleotide-based immunogens according to the methods of the invention (an initial administration).
- an initial administration the immune response may be supplemented or further enhanced by one or more subsequent administrations of said immunogens, where said subsequent administration occurs at about two weeks after the initial administration, at about four weeks after the initial administration, at about eight weeks after the initial administration, or within about one month after the initial administration, or within about two months after the initial administration, or within about six months of the initial
- epitope refers to an immunogenic amino acid sequence.
- An epitope may refer to a minimum amino acid sequence of typically 6-8 amino acids which minimum sequence is immunogenic when removed from its natural context, for example when transplanted into a heterologous polypeptide.
- An epitope may also refer to that portion of a protein which is immunogenic, where the polypeptide containing the epitope is referred to as the antigen (or sometimes "polypeptide antigen").
- a polypeptide or antigen may contain one or more (e.g., 2 or 3 or more) distinct epitopes.
- epitope embraces B-cell and T-cell epitopes.
- T-cell epitope embraces CD4+ T-cell epitopes and CD8+ T-cell epitopes (sometimes also referred to as CTL epitopes).
- immunogenic polypeptide refers to a polypeptide which is immunogenic, that is to say it is capable of eliciting an immune response in an animal, and therefore contains one or more epitopes (e.g., T-cell and/or B-cell epitopes). Immunogenic polypeptides may contain one or more epitopes, and these may be in a natural or an unnatural arrangement, including epitopes from different polypeptides, as in a fusion protein.
- Immunogenic polypeptides will typically be recombinant proteins produced, e.g., by expression in a heterologous host such as a bacterial host, in yeast or in cultured mammalian cells.
- the immunogenic polypeptide comprises at least one T cell epitope.
- the immunogenic polypeptide further comprises at least one B cell epitope.
- Immunogenic polypeptides used in the present invention may be in the form of immunogenic derivatives or immunogenic fragments of immunogenic polypeptides.
- immunogenic derivative in relation to an immunogenic polypeptide of native origin refers to an antigen modified in a limited way relative to its native counterpart.
- the immunogenic derivative may include a point mutation which may change the properties of the protein relative to the native counterpart, e.g. by improving expression in prokaryotic systems or by removing undesirable activity, e.g. enzymatic activity.
- Immunogenic derivatives will however be sufficiently similar to the native antigens such that they retain their antigenic properties and remain capable of raising an immune response against the native antigen.
- an "immunogenic fragment" of a polypeptide is a fragment which encodes at least one epitope of the polypeptide, for example a cytotoxic T lymphocyte (CTL) epitope, and is typically at least 8 amino acids in length.
- CTL cytotoxic T lymphocyte
- Fragments of at least 8, for example 8 to 10 amino acids or up to 20, 50, 60, 70, 100, 150 or 200 amino acids in length are considered to fall within the scope of the invention as long as the polypeptide demonstrates antigenicity, that is to say that the major epitopes (e.g., CTL epitopes) are retained by the polypeptide fragment.
- major epitopes e.g., CTL epitopes
- polynucleotide-based immunogens are those delivered by a nucleic acid encoding a polypeptide immunogen.
- Polynucleotide-based immunogens comprise expression vectors that enter cells and express the polypeptide immunogen.
- the terms refers to both nucleic acids administered as a purified form, e.g., an expression plasmid, as well as nucleic acids that are administered as a virus (within a viral capsid).
- the nucleic acid encodes immunogenic epitopes from an antigen of interest that induces humoral and/or cellular immune responses.
- the nucleic acid component can be any nucleic acid, including DNA or RNA.
- the nucleic acid component can be a viral RNA or a messenger RNA.
- nucleic acid expression systems such as plasmid DNA, bacterial and viral expression systems.
- polynucleotide sequence encoding the immunogen may be codon optimized for expression in mammalian cells. Desirably the codon usage pattern of the polynucleotide sequence is typical of highly expressed human genes. The principle of such codon optimization is described, for example, in WO 05/025614. When several antigens or epitopes are provided as a fusion protein, such fusion would be encoded by a polynucleotide under the control of a single promoter.
- DNA vaccines may comprise a bacterial plasmid vector into which is inserted a viral promoter, a sequence encoding the antigenic peptide to be delivered, and polyadenylation/transcriptional termination sequences.
- the sequence may encode a full protein, a fusion protein comprising different antigens, or simply an antigenic peptide sequence against which an immune response is desired.
- the plasmid is taken up by cells of the host (e.g., a vaccinated subject) where the encoded peptide is produced.
- Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the host
- the system for expressing the immunogen may be a recombinant live microorganism, such as a virus or bacterium into which the sequence expressing the immunogen
- viral vectors used in the present invention comprise one or more heterologous polynucleotides which encode one or more immunogenic polypeptides.
- the viral vector may be any suitable viral vector.
- Viruses useful for expressing an immunogen include poxviruses (e.g; vaccinia, fowlpox, canarypox, modified poxviruses e.g.
- VMA Modified Virus Ankara
- Japanese encephalitis virus adenoviruses, adeno-associated virus, picomaviruses (poliovirus, rhinovirus), herpesviruses (varicella zoster virus, etc). These viruses can be attenuated in various ways in order to obtain live vaccines.
- the adenoviruses are a large family of double-stranded DNA viruses. Wold &
- Adenovirus methods and protocols Adenoviruses, Ad Vectors, Quantitation, and
- Virus types include: dsDNA viruses (e.g. Adenoviruses, Herpesviruses, Poxviruses); ssDNA viruses (+) sense DNA (e.g. Parvoviruses); dsRNA viruses (e.g. Reoviruses);
- dsDNA viruses e.g. Adenoviruses, Herpesviruses, Poxviruses
- ssDNA viruses (+) sense DNA e.g. Parvoviruses
- dsRNA viruses e.g. Reoviruses
- (+)ssRNA viruses (+) sense RNA e.g. Picomaviruses, Togaviruses
- sense RNA e.g. Picomaviruses, Togaviruses
- sense RNA e.g. Picomaviruses, Togaviruses
- sense RNA e.g. Picomaviruses, Togaviruses
- sense RNA e.g. Picomaviruses, Togaviruses
- RNA e.g. Orthomyxoviruses, Rhabdoviruses
- ssRNA-RT viruses (+) sense RNA with DNA intermediate in life-cycle e.g. Retroviruses
- dsDNA-RT viruses e.g. Hepadnaviruses
- DNA virus types include: Adenoviridae; Papillomaviridae; Parvoviridae; Herpesviridae e.g. Herpes simplex virus, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus;
- Poxyiridae e.g. Smallpox virus, vaccinia virus; Hepadnaviridae e.g. Hepatitis B virus;
- Polyomaviridae e.g. Polyoma virus, JC virus (progressive multifocal leucoencephalopathy);
- Circoviridae e.g. Transfusion Transmitted Virus.
- RNA virus types include Reoviridae e.g.
- Reovirus Rotavirus
- Picornaviridae e.g. Enterovirus, Rhinovirus, Hepatovirus, Cardiovirus
- Caliciviridae e.g. Norwalk virus, Hepatitis E; Togaviridae e.g. Rubella virus; Arenaviridae e.g.
- Lymphocytic choriomeningitis virus Flaviviridae e.g. Dengue virus, Hepatitis C virus, Yellow fever virus; Orthomyxoviridae e.g. Influenzavirus A, Influenzavirus B, Influenzavirus C, Isavirus, Thogotovirus; Paramyxoviridae e.g. Measles virus, Mumps virus, Respiratory syncytial virus; Bunyaviridae e.g. California encephalitis virus, Hantavirus; Rhabdoviridae e.g. Rabies virus; Filoviridae e.g. Ebola virus, Marburg virus; Coronaviridae e.g. Corona virus; Astroviridae e.g.
- Astrovirus Bornaviridae e.g. Borna disease virus.
- RT virus types include Metaviridae; Pseudoviridae; Retroviridae— e.g. HIV; Hepadnaviridae— e.g. Hepatitis B virus; Caulimoviridae— e.g. Cauliflower mosaic virus.
- the vector is the measles virus.
- Measles virus belongs to the genus Morbillivirus in the family Paramyxoviridae.
- Measles viral vectors are disclosed in, by way of example, WO2008/078198, WO 2006/136697, WO2004/001051 and WO2004/000876, Combredet et al., Journal of Virology 77 (21): 11546-11554 (2003).
- the vector is selected from the adenoviral vectors described in PCT/EP2010 000616 (published as WO 2010/086189 and US 2012-0027788 ) and PCT/EP2009/000672 (published as WO 2010/085984). In one aspect of the invention the vector is selected from the replication defective viral vectors ChAd83 and PanAd3.
- the nucleic acid expression system is administered in sufficient amounts to transduce the target cells and provide sufficient levels of gene transfer and expression and to permit specific immune responses to the administered immunogen to develop. Such dosages will vary depending upon the immunogen administered, the condition being treated, the subject being treated, and other factors, and can be determined by those skilled in the medical arts.
- polynucleotide or polynucleotides encoding immunogenic polypeptides to be expressed may be inserted into any suitable region of the viral vector, for example into a deleted region. Although two or more polynucleotides encoding immunogenic polypeptides may be linked as a fusion, the resulting protein may be expressed as a fusion protein, or it may be expressed as separate protein products, or it may be expressed as a fusion protein and then subsequently broken down into smaller subunits.
- the viral vector is suitably replication competent in the host organism to which it is to be delivered.
- the viral vector is attenuated or replication defective (unable to replicate in mammalian hosts, e.g., Fowlpoxvirus (FPV) and modified vaccinia virus Ankara strain (MVA)).
- FPV Fowlpoxvirus
- MVA modified vaccinia virus Ankara strain
- the viral vector of the invention may be replication defective. This means that it has a reduced ability to replicate in non-complementing cells, compared to the wild type virus. This may be brought about by mutating the virus e.g. by deleting a gene involved in replication.
- the viral vector is not affected by, or only minimally affected by the presence of an adjuvant. In one aspect any reduction in viral titer caused by the adjuvant is no more than 50%, such as no more than 40%, 30%, 20%, 15%, 10%, 5% and in a further aspect there is no reduction in titer at all.
- the methods of the present invention may be used to deliver therapeutic anti-cancer vaccines.
- Antigen-based immunotherapy to treat cancer utilizes tumor-associated antigens to elicit specific host immune responses against the antigen, in order to train the patient's immune system to eliminate tumor cells.
- a tumor-associated antigen is a protein frequently expressed by a tumor type and capable of inducing an immune response in the subject. Certain tumor-associated antigens are expressed selectively or preferentially on cancer cells. Tumor antigens can be classified into 5 groups: (1) cancer-testis antigens that are expressed in a range of different tumor types but not in normal tissues except testis (e.g., MAGE and NY-ESO-1), (2) melanocyte differentiation antigens expressed in melanoma and normal melanocytes (e.g.
- antigens encoded by mutated normal gene e.g. p53 and ras
- antigens encoded by mutated normal gene e.g. p53 and ras
- self-antigens overexpressed in malignant tissues e.g. HER-2/ neu
- antigens derived from oncogenic viruses e.g. HPV and EBV.
- tumor-associated antigens including the cancer-testis antigens of the B AGE, GAGE, MAGE, NY-ESO- 1 , PRAME and S SX families; differentiation antigens (including GP100, Mel an- A/Mart- 1, PSA, CEA, and Mammaglobin-A); antigens overexpressed in tumor tissues (including p53, her2/neu, survivin, WT1); tumor-associated carbohydrate antigens (including Muc-1).
- tumor cells from a single tumor, or within a single subject may be heterogeneous, that is, with some tumor cells expressing the tumor-associated antigen while other cells do not.
- Such tumors are, for purposes of the present invention, considered as tumor antigen-expressing tumors.
- tumor-associated antigens are referred to by way of example, and are not intended to be limiting upon the scope of the present invention.
- MAGE-A3 is a member of the MAGE family.
- MAGE-A3 antigenic polypeptides and fusion proteins are in clinical trials as anti-cancer vaccine antigens. See, e.g., Vansteenkiste et al., J. Clin. Oncol 31 :2396-403 (2013); Kruit et al., J. Clin Oncol. 31 :413-20 (2013); Kruit et al., Int. J. Cancer 117:596-604 (2005); Marchand et al., Eur. J. Cancer 39:70-77 (2003); Atanackovic et al., Proc. Natl. Acad. Sci. 105: 16-50-1655 (2008); Vantomme et al., J.
- the human MAGE-A3 sequence is known in the art, see e.g., GenBank Accession CAG46573, NCBI Reference Sequence NP_005353.1 :
- recMAGE-A3 polypeptide immunogen refers to a specific 450-amino acid recombinant protein having SEQ ID NO:2.
- recMAGE-A3 contains a Protein D sequence (amino acids 1-127 of SEQ ID NO:2), inserted Met- Asp (amino acids 128-129 of SEQ ID NO:2)
- MAGE- A3 -specific antibodies may not directly be involved in removing tumor cells, they may contribute to CD8 + T-cell cytotoxic activity because antibody-mediated opsonization of MAGE- A3 has been found to promote cross-presentation to naive T cells. Moeller et al., Cancer Immunol. Immunother. 61 :2079 (2012).
- a MAGE-A3 immunogenic polypeptide and a polynucleotide-based vector encoding a MAGE-A3 immunogenic polypeptide are provided.
- Such administration may occur after surgical, chemotherapeutic, radiologic, or other treatment of the tumor, including in cases where, subsequent to such treatment, there is no evidence of disease.
- An adjuvanted recombinant MAGE- A3 fusion polypeptide of SEQ ID NO:2 has been used in clinical trials for the therapy of cancer, including where the adjuvant was AS02B or AS 15, as described herein.
- Clinical trials of adjuvanted, recombinant MAGE- A3 fusion polypeptide have included subjects with Non-small cell lung cancer (NSCLC), melanoma, and bladder cancer. See, e.g., Peled et al., Immunotherapy 1(1): 19-25 (209).
- one embodiment of the present invention is the co-administration of an immunogenic polypeptide having SEQ ID NO:2 and a polynucleotide-based immunogen (such as a viral vector) encoding a full length MAGE A3 polypeptide (such as a polypeptide of SEQ ID NCv l).
- a polynucleotide-based immunogen such as a viral vector
- a full length MAGE A3 polypeptide such as a polypeptide of SEQ ID NCv l
- adjuvant refers to a composition that enhances the immune response to an immunogen.
- Adjuvants are described in general, e.g. in Vaccine Design—the Subunit and Adjuvant Approach, Powell and Newman, Plenum Press, New York, 1995.
- An immune response is generated to an antigen through the interaction of the antigen with the cells of the immune system.
- the resultant immune response may be broadly classified into two categories: the humoral and cell mediated immune responses (traditionally
- Thl-type responses cell-mediated response
- Th2- type immune responses humoral response
- Thl-type immune responses may be characterized by the generation of antigen specific, haplotype restricted cytotoxic T lymphocytes, and natural killer cell responses.
- Thl-type responses are often characterised by the generation of antibodies of the IgG2a subtype, whilst in the human these correspond to IgGl type antibodies.
- Th2-type immune responses are characterised by the generation of a broad range of immunoglobulin isotypes including in mice IgGl, IgA, and IgM.
- Thl and Th2-type immune responses is not absolute. In reality an individual will support an immune response which is described as being predominantly Thl or predominantly Th2.
- Th2-type cytokine responses Traditional the best indicators of the Thl :Th2 balance of the immune response after a vaccination or infection includes direct measurement of the production of Thl or Th2 cytokines by T lymphocytes in vitro after re-stimulation with antigen, and/or the measurement of the IgGl :IgG2a ratio of antigen specific antibody responses.
- the nature of the T-cell response to a vaccine immunogen can be influenced by the composition of the adjuvant used in the vaccine. For instance, adjuvants containing MPL & QS21 have been shown to activate Thl CD4+ T cells to secrete IFN-gamma (Stewart et al. Vaccine. 2006, 24 (42- 43):6483-92).
- Thl-type adjuvant is one which stimulates isolated T-cell populations to produce high levels of Thl-type cytokines when re-stimulated with antigen in vitro, and induces antigen specific immunoglobulin responses associated with Thl-type isotype.
- MPL monophosphoryl lipid A
- 3D-MPL 3-de-O-acylated monophosphoryl lipid A
- 3D-MPL 3-de-O-acylated monophosphoryl lipid A
- It can be purified and prepared by the methods taught in GB 2122204B, which reference also discloses the preparation of diphosphoryl lipid A, and 3-O-deacylated variants thereof.
- Other purified and synthetic lipopolysaccharides have been described (U.S. Pat. No. 6,005,099 and EP 0 729 473 B l;
- Saponins are also known Thl immunostimulants. Lacaille-Dubois, M and Wagner H, A review of the biological and pharmacological activities of saponins. Phytomedicine vol 2 pp 363-386 (1996).
- saponin Quil A derived from the bark of the South American tree Quillaja Saponaria Molina
- Purified fractions of Quil A are also known as immunostimulants, such as QS21 and QS17; methods of their production is disclosed in U.S. Pat. No.
- QS7 a non-haemolytic fraction of Quil-A
- Use of QS21 is further described in Kensil et al. (1991, J. Immunology, 146: 431-437).
- Combinations of QS21 and polysorbate or cyclodextrin are also known (WO 99/10008).
- Particulate adjuvant systems comprising fractions of QuilA, such as QS21 and QS7 are described in WO 96/33739 and WO 96/11711.
- CpG immunostimulatory oligonucleotide containing unmethylated CpG dinucleotides
- CpG is an abbreviation for cytosine-guanosine dinucleotide motifs present in DNA.
- CpG is known as an adjuvant when administered by both systemic and mucosal routes (WO 96/02555, EP 468520, Davis et al, J.Immunol, 1998, 160:870-876; McCluskie and Davis, J.Immunol., 1998, 161 :4463- 6).
- CpG when formulated into vaccines, may be administered in free solution together with free antigen (WO 96/02555; McCluskie and Davis, supra) or covalently conjugated to an antigen (WO 98/16247), or formulated with a carrier such as aluminium hydroxide (Davis et al, supra ; Brazolot-Millan et al., Proc. Natl. Acad. Sci., USA, 1998, 95: 15553-8).
- free antigen WO 96/02555; McCluskie and Davis, supra
- WO 98/16247 covalently conjugated to an antigen
- a carrier such as aluminium hydroxide
- Immunostimulants such as those described above may be formulated together with carriers, such as liposomes, oil in water emulsions, and or metallic salts (including aluminum salts such as aluminum hydroxide).
- carriers such as liposomes, oil in water emulsions, and or metallic salts (including aluminum salts such as aluminum hydroxide).
- 3D-MPL may be formulated with aluminum hydroxide (EP 0 689 454) or oil in water emulsions (WO 95/17210);
- QS21 may be formulated with cholesterol containing liposomes (WO 96/33739), oil in water emulsions (WO 95/17210) or alum (WO 98/15287);
- CpG may be formulated with alum (Davis et al, supra ; Brazolot- Millan, supra) or with other cationic carriers.
- Combinations of immunostimulants may be utilized in the present invention, in particular a combination of a monophosphoryl lipid A and a saponin derivative (see, e.g., WO 94/00153; WO 95/17210; WO 96/33739; WO 98/56414; WO 99/12565; WO 99/11241), more particularly the combination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a composition where the QS21 is quenched in cholesterol-containing liposomes (DQ) as disclosed in WO 96/33739.
- a combination of CpG plus a saponin such as QS21 is an adjuvant suitable for use in the present invention.
- a potent adjuvant formulation involving QS21, 3D-MPL & tocopherol in an oil in water emulsion is described in WO 95/17210 and is another formulation for use in the present invention.
- Saponin adjuvants may be formulated in a liposome and combined with an immunostimulatory oligonucleotide.
- suitable adjuvant systems include, for example, a combination of monophosphoryl lipid A, preferably 3D-MPL, together with an aluminium salt (e.g. as described in WO00/23105).
- a further exemplary adjuvant comprises comprises QS21 and/or MPL and/or CpG.
- QS21 may be quenched in cholesterol-containing liposomes as disclosed in WO 96/33739.
- Immunostimulant AS02B is a combination of QS21 saponin and monophosphoryl lipid A (a TLR-4 agonist) in an oil/water emulsion.
- Immunostimulant AS 15 is a combination of QS21, monophosphoryl lipid A, and CpG7909 (a TLR-9 agonist), in a liposomal formulation. Kruit et al. (2013) concluded that clinical activity seemed to be higher when recMAGE- A3 was used with AS 15.
- Suitable adjuvants include alkyl Glucosaminide phosphates (AGPs) such as those disclosed in WO9850399 or U.S. Pat. No. 6,303,347 (processes for preparation of AGPs are also disclosed), or pharmaceutically acceptable salts of AGPs as disclosed in U.S. Pat. No. 6,764,840.
- AGPs alkyl Glucosaminide phosphates
- Some AGPs are TLR4 agonists, and some are TLR4 antagonists. Both are thought to be useful as adjuvants.
- the adjuvant component does not contain any virus.
- the adjuvant component does not contain any virus.
- compositions for use according to the invention do not contain any virus other than the one or more more viral vectors comprising one or more heterologous polynucleotides encoding one or more immunogenic polypeptides.
- compositions The amount of adjuvant will depend on the nature of the adjuvant and the immunogenic polypeptide, the condition being treated and the age, weight and health of the subject.
- compositions administered as part of the methods described herein may be formulated for pharmaceutical administration.
- Carriers used in pharmaceutical compositions will vary, as is known in the art, depending upon the route and mode of administration. Subjects
- the present methods of immunization are suitable for use in mammalian subjects, including humans, non-human primates, small mammals including rodents (mice and rats, e.g.) and rabbits, and domesticated animals such as cattle, horses, sheep, dogs and cats.
- the immunogenic compositions are co-administered to subjects in amount(s) sufficient to elicit an immune response , e.g., a CD8, CD4, and/or antibody response to the antigen of interest to which the nucleic acid/protein components are directed.
- an immune response e.g., a CD8, CD4, and/or antibody response to the antigen of interest to which the nucleic acid/protein components are directed.
- the amount of immunogenic compositions are sufficient to induce a therapeutic immune response, e.g., a response that at least partially arrests or slows symptoms, progression, and/or complications of a disease, e.g. cancer.
- An amount adequate to accomplish this is defined as "therapeutically effective dose.”
- the amount of immunogenic compositions are sufficient to induce a prophylactic immune response, e.g., a response that at least partially reduces the incidence or prevalence of disease.
- a prophylactic immune response e.g., a response that at least partially reduces the incidence or prevalence of disease.
- An amount adequate to accomplish this is defined as a "prophylactically effective dose.”
- Amounts effective to achieve therapeutic or prophylactic immune responses will depend on, e.g., the particular composition of the regimen administered, the manner of administration, the stage and severity of the disease, the general state of health of the patient, and the judgment of the prescribing health care provider.
- Assessment of Immunogenic Response will depend on, e.g., the particular composition of the regimen administered, the manner of administration, the stage and severity of the disease, the general state of health of the patient, and the judgment of the prescribing health care provider.
- Measurements to evaluate vaccine response include but are not limited to: antibody measurements in the plasma, serum, saliva, or other body fluids; analysis of in vitro cell proliferation in response to a specific antigen, indicating the function of CD4' cells; analysis of cytokine production of lymphocytes after stimulation with the specific antigen or with pools of peptides of the specific antigen; and analysis ofneutralizing activity found in the serum or plasma using virus inhibition as says well known in the art.
- Such assays are well known in the art.
- Other measurements of immune response include assessing CD8+ responses, for example, using tetramer staining of fresh or cultured PBMC (see, e.g., Altman, et al. , Proc. Natl. Acad. Sci. USA 90: 10330, 1993; Altman, et al. , Science 274:94, 1996), or 7-interferon release assays such as ELISPOT assays (see, e.g., Lalvani, et al. , J. Exp. Med. 186:859, 1997; Dunbar, et al. , Curr. Biol. 8:413, 1998; Murali-Krishna, et al. , Immunity 8: 177, 1998), or by using functional cytotoxicity assays.
- ELISPOT assays see, e.g., Lalvani, et al. , J. Exp. Med. 186:859, 1997; Dunbar, et al
- the antibody response is measured.
- kits comprising the polynucleotide and polypeptide components to be administered in accordance with the methods described herein.
- a kit can comprise for example, a container that includes one or more of the
- kits of the invention can comprise a polypeptide form of the antigen separate from the polynucleotide form of the antigen, and may comprise a separate adjuvant.
- the kit may also include additional components, e.g., for mixing with one or both of the compositions before administration, such as diluents, carriers, and the like.
- peptide, ""polypeptide, “ and “protein” are used interchangeably herein to refer to at least two amino acids or amino acid analogs that are covalently linked by a peptide bond or an analog of a peptide bond.
- the amino acids of the peptide may be L-amino acids or D-amino acids.
- a peptide, polypeptide or protein may be synthetic, recombinant or naturally occurring.
- a synthetic peptide is a peptide produced by artificial means in vitro.
- the terms “identical” in the context of two or more nucleic acids or two or more polypeptide sequences refers to two or more sequences that have the same nucleotide or amino acids sequence, respectively.
- the terms “similar” or “substantially similar” or “substantially the same” refer to sequences having a specified percentage of amino acid residues or nucleotide residues that are the same (ie., about 70% identity, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region of an antigen of interest or a nucleic acid encoding an antigen of interest when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms, or by manual alignment and visual inspection (see, e.g., NCBI web site or the like).
- the definition also includes sequences that have deletions
- BLAST Altschul et al. , Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al. , J.Mol. Biol. 215:403-410(1990),respectively.
- BLAST software is publicly available through the National Center for Biotechnology Information, e.g., on the worldwide web..
- enhanced immune response or “increased immune response” as used herein refers to an immune response that includes increases in the level of immune cell activation, and/or an increase in the duration of the response, and/or improved immunological memory, and/or an improvement in the kinetics of the immune response. Such increases can be demonstrated by either a numerical increase, e.g., an increased in levels of antibody in a particular time frame, as assessed in an assay to measure the response assay, or by prolonged longevity of the response.
- Antigen is used herein as synonymous with immunogen, and antigenic is used herein as synonymous with immunogenic.
- homolateral or ipsilateral administration refers to administration in the limbs on one side of a subject (i.e., administrations in the left arm and left leg of a subject are considered as ipsilateral administration).
- contralateral administration means administration in limbs on the opposite sides of a subject (e.g., separate administrations in the left arm and the right arm of a subject are considered to be contralateral administration, as are separate administrations in the left leg and right leg of a subject). Administrations to the same limb of a subject are considered to be at the same anatomic site.
- immunogen-specific CD4+ T-cells and/or CD8+ T-cells and/or antibodies is meant CD4+ T-cells and/or CD8+ T-cells and/or antibodies which specifically recognise a specific immunogen or part thereof (e.g., an epitope thereof).
- specifically recognise is meant that said CD4+ T-cell and/or CD8+ T-cell recognition is immunospecific rather than a non-specific.
- recMAGE-A3 AS-15 adjuvanted protein refers to a composition comprising the recMAGE-A3 polypeptide of SEQ ID NO:2 and AS 15 adjuvant.
- AS 15 Adjuvant System 15
- PanAd3-MAGEA3 refers to the chimpanzee viral vector PanAd3 (as described in PCT/EP/2010/000616 (published as WO 2010/086189) see also EP10702615 and US 13/147, 193) expressing a full-length MAGE-A3 protein of SEQ ID NO: l .
- MVA-MAGEA3 refers to a Modified Vaccinia Ankara viral vector expressing a full-length MAGE- A3 protein of SEQ ID NO: 1.
- MVA is a highly attenuated strain of vaccinia virus; see, e.g., Mayr et al., Monbl Bakteriol. B. 167(5-6):375-90 (1978) and Mayr et al., Infection 3 :6-14 (1975).
- Example 1 Materials & Methods for Comparison of Co-administered schedules in Mice
- mice Female CB6F1 mice (six weeks old) were purchased from Harlan Laboratories.
- Group 1 and 2 received, respectively, PanAd3-MAGEA3 vector given in one site (25 ⁇ corresponding to 5xl0 6 vp) or given contralaterally (25 ⁇ per injection corresponding to 2.5 xl0 6 vp each injection for a total dose of 5xl0 6 vp).
- Groups 3 and 4 received the co-administration of Pan Ad3 -MAGE A3 and recMAGEA3
- Group 6 was injected with only 25 ⁇ recMAGEA3 AS 15 -adjuvanted protein ( ⁇ ⁇ ).
- one site refers to injection in the same limb.
- the vaccination schedule was: intramuscular (EVI) administration ⁇ at week zero(0), with analysis at two (2) and four (4) weeks post administration.
- Group 1 PanAd3 -MAGE (at single injection site)
- Group 2 PanAd3-MAGE (at two contralateral injection sites)
- Group 4 PanAd3-MAGE + recMAGE-A3 AS-15 adjuvanted protein (PanAd3-Mage and recMAGE-A3 AS-15 adjuvanted protein injected separately, at same site)
- Group 5 PanAd3-MAGE + recMAGE-A3 AS-15 adjuvanted protein (PanAd3-Mage and recMAGE-A3 AS-15 adjuvanted protein co-formulated into a single injection)
- ASCI Antigen Specific Cancer Immunotherapy and in the Tables herein, refers to recMAGE-A3 AS- 15 adjuvanted protein.
- Example 2 Materials & Methods for MAGEA3 prime/boost with viral vectors and ASCI
- the immunogenicity of sequential (prime-boost) administrations of polynucleotide vectors and polypeptide immunogens was evaluated, using different administration schedules.
- mice Female CB6F1 mice (six weeks old) were purchased from Harlan Laboratories;
- mice were all primed intramuscularly (in the quadriceps) with Pan Ad3 -MAGE A3 alone in a volume of 50 ⁇ 1 corresponding to 5xl0 6 vp (Groups 5-6-7), or with using concomitant contralateral administration of Pan Ad3 -MAGE A3 and 50 ⁇ 1 recMAGEA3 AS 15 -adjuvanted protein corresponding to ⁇ g (Groups 1-2-3).
- Control group (Group 4) was primed with 50 ⁇ 1 of recMAGEA3 AS 15 -adjuvanted protein corresponding to ⁇ g.
- mice Seven groups of CB6F1 mice (five mice in each group) were used. Each group was vaccinated three times: at Week Zero [prime], Week Four [second immunization or first boost] and Week 8 [third immunization or second boost].
- ASCI Antigen Specific Cancer Immunotherapy and, in the Tables herein, refers to recMAGE- A3 AS- 15 adjuvanted protein.
- IFNy ELISpot IFN-y-producing T cells were evaluated by the following ELISPOT assay, in Experiment 1 at four weeks post vaccination, and in Experiment 2 at week 10 (two weeks post second boost).
- MSIP S4510 plates (Millipore) were coated with lC ⁇ g/ml of anti-mouse (U-CyTech Utrecht, The Netherlands) overnight at 4°C. After washing and blocking, mouse splenocytes were plated in duplicate at two different densities (2 x 10 5 and 4 x 10 5 cells/well) and stimulated overnight with CD8 MAGEA3 immunodominant peptide or with a pool of MAGE- A3 overlapping 15mer peptides at final concentration of ⁇ g/ml each peptide.
- peptide diluent DMSO Sigma- Aldrich, Milan, Italy
- Concanavalin A polypeptide Sigma- Aldrich, Milan, Italy
- Biotinylated anti-mouse IFNy antibody U-CyTech Utrecht, The Netherlands
- Streptavidin-Alkaline Phosphatase conjugated BD Biosciences, NJ
- BCIP/NBT 1-Step solution Thermo Fisher Scientific, Rockford, IL. Plates were acquired and analyzed by an A.EL.VIS (Automated ELisa-spot assay Video System, Hannover, Germany) automated plate reader.
- ELISpot response was considered positive when all of the following conditions were met: IFNy production present in Con-A stimulated wells; at least 50 specific spots/million splenocytes present; the number of spots seen in positive wells was three times higher than the number detected in the mock control wells (DMSO); and decreased responses with cell dilutions were seen. ELISpot data were expressed as IFNy spot forming cells (SFC) per million splenocytes.
- mice splenocytes were stimulated at 37°C in 5% C0 2 for 15-20 hours using a pool of overlapping 15-mer MAGEA3 peptides as antigen at a final concentration of 2 ⁇ g/ml each peptide in the presence of Golgi-plug (according to the manufacturer's
- DMSO Sigma-Aldrich, Milan, Italy
- PMA/Ionomycin Sigma-Aldrich, Milan, Italy
- mouse splenocytes were incubated with purified anti-mouse CD16/CD32 clone 2.4G2 (Fc block: BD Biosciences, NJ) and then stained in FACS buffer (PBS, 1% FCS) with the following surface antibodies: APC anti-mouse CD3e, clone 145-2C11; PE anti-mouse CD4, clone L3T4; PerCP anti-mouse CD8a, clone 53-6.7 (all from BD Biosciences, NJ).
- FACS buffer PBS, 1% FCS
- Intracellular staining was performed after treatment with Cytofix/Cytoperm and in the presence of PermWash (BD Biosciences, NJ) using FITC anti-mouse IFNy, clone XMG1.2 (BD
- CD8-Antigen specific T cells by MAGEA3 pentamer staining Analysis of CD 8 -antigen specific T cells was performed by the following pentamer staining method, in Experiment 1 using individual blood samples taken from mice at two weeks and four weeks post vaccination; and for Experiment 2 using blood samples taken at week two, week four (pre-boost), week 5, week 8, week 9, and week 10 (pooled blood samples from each group were tested, except for samples taken at week 5 and week 9 when individual samples were tested).
- Blood (about 400 ⁇ ) was taken from each mouse via the retro-orbital sinus with a Pasteur pipette and collected in tubes containing Heparin as anticoagulant. Two steps of red blood cell lysis were performed incubating the samples with ACK lysing buffer (Gibco) for 15 minutes.
- ACK lysing buffer Gibco
- Results were obtained by pentamer staining on whole blood at week 2 and week 4 (individual mice, shown in Figure 1) and IFNy ELISpot-ICS on spleen at week 4 (individual mice, Figure 2).
- the percentage of MAGEA3 -specific CD8 cells is much greater at both time points in Group 3 (contralateral co-administration of PanAd3-MAGEA3 and recMAGEA3 AS15-adjuvanted protein) compared to co-administration in the same limb of Pan Ad3 -MAGE A3 and recMAGEA3 AS15-adjuvanted protein (Group 4), and to co-formulated coadministration of Pan Ad3 -MAGE A3 and recMAGEA3 AS15-adjuvanted protein (Group 5).
- Figure 2 shows the % of MAGEA3 specific CD4 cells as well as CD8 cells, measured at week 4 post injection. (This analysis was not done for Groups 5 and 6).
- MAGEA3 specific CD8 responses are shown in Figure 3, where Groups 1-7 are shown from left to right across the graph. Data was generated with pooled blood samples from each group, except at week 5 and week 9 where individual samples were tested (Mean + SEM shown).
- MAGEA3 specific CD8 responses are shown in Figure 4. Circles represent CD4 cells and squares represent CD8 cells; a horizontal line represents the geometric mean for each cell type per group. The data indicate that regimens based on contralateral administration of viral vector and polypeptide immunogen induced potent CD4 and CD8 T cell responses. Sequences
- SEQ ID NO: l human MAGE- A3 protein
- SEQ ID NO:2 (recMAGEA3; fusion protein of Protein D fragment + MAGEA3 amino acids 3- 314)
- SEQ ID NO:4 (CD 8 MAGEA3 immunodominant peptide) : SYVKVLHHM
Abstract
A polypeptide immunogen for use in a method of raising an immune response, comprising the co-administration of an immunogenic polypeptide and a polynucleotide encoding a polypeptide immunogen, where the agents are administered concomitantly and at different anatomic sites of an individual subject in need of treatment. The methods are capable of inducing CD4+ and CD8+ T-cell responses as well as antibody responses in subjects, without recourse to complex sequential prime-boost schedules.
Description
CONTRALATERAL CO-ADMINISTRATION OF VACCINES
TECHNICAL FIELD
The present invention relates to a method of raising an immune response against a polypeptide antigen, comprising the co-administration of an immunogenic polypeptide and a polynucleotide encoding a polypeptide immunogen, where the agents are administered concomitantly (co-administered) and at different anatomic sites of an individual subject in need of treatment. The methods are capable of inducing CD4+ and CD8+ T-cell responses as well as antibody responses in subjects, without recourse to complex sequential prime-boost schedules.
BACKGROUND
Methods of vaccinating to raise an immune response include the administration of a protein or polypeptide which stimulates an aspect of the immune response in vivo, as well as the administration of a polynucleotide which, once properly administered to a subject, is transcribed and translated into a protein or polypeptide which stimulates an aspect of the immune response in vivo.
Prime-boost vaccination is a strategy for eliciting an immune response to a target antigen (the immunogen), which can use multiple methods of administering the antigen. (Woodland, Jump-starting the immune system: prime-boosting comes of age. Trends Immunol 25: 98-104 (2004)). One administration of the antigen (by a first method) is considered to 'prime' the immune system; the administration of the same antigen (by a different method) is considered to 'boost' the immune response. Prime-boost vaccination has been shown to be capable of inducing high levels of antigen-specific CD4+ and CD8+ cells. In one approach, the 'priming' administration and the 'boosting' administration occur sequentially in time. The administration may occur at the same anatomic site (e.g., the same limb) or at separate anatomic sites (e.g., contralateral^, using left and right limbs; or ipsilaterally using sites on different limbs).
Heterologous prime-boost protocols may, for example, comprise administration of a
pharmaceutical composition comprising a polypeptide immunogen, and administration of a pharmaceutical composition comprising a polynucleotide that expresses a polypeptide immunogen.
In addition to prime-boosting vaccination regimes where the administration of a polypeptide-based immunogen is alternated (separated in time) with the administration of polynucleotide-vector based vaccines, methods of co-administration (concomitant
administration) of polypeptide-based and polynucleotide-based immunogens have been described. Published US patent applications US 2011 0236468 and US 2010 0055166 describe
methods of vaccination by the concomitant administration of (a) a first immunogenic polypeptide derived from a pathogen, (b) a viral vector comprising a heterologous
polynucleotide encoding a second immunogenic polypeptide derived from the same pathogen; and (c) an adjuvant.
The mammalian immune response has two key components: the humoral response and the cell-mediated response. The humoral response involves the generation of circulating antibodies which bind to the antigen to which they are specific, thereby neutralising the antigen and initiating its clearance by cells of the immune system. The cell-mediated response involves the interplay of numerous different types of cells, including T cells. T cells are divided into subsets, including the CD4+ and CD8+ T cells.
To induce an optimal immune response for either prophylactic or therapeutic purposes, stimulation of both CD4+ and CD8+ cells is desirable. This is one of the main goals of "prime- boost" vaccination strategies in which the administration of polypeptide-based vaccines (inducing mostly CD4+ T cells) is alternated (separated in time) with the administration of polynucleotide-vector based vaccines, e.g.. naked DNA, viral vectors or intracellular bacterial vectors such as Listeria, (inducing mostly CD8+ T cells). However, prime-boost vaccine strategies require multiple vaccinations and can be burdensome or unviable, especially in mass immunization programs or programs in the developing world.
SUMMARY OF THE INVENTION
An aspect of the present invention is a method of raising an immune response in a subject, by administering (a) a polypeptide immunogen and (b) a polynucleotide vector encoding the same polypeptide immunogen, where the polypeptide immunogen and the polynucleotide-based vector are administered at about the same time, in different limbs of the subject.
A further aspect of the present invention is a method of treating cancer in a subject in need of such treatment, by administering (a) a polypeptide immunogen selected from a tumor- associated antigen, an immunogenic fragment of a tumor-associated antigen, and a fusion protein comprising a tumor-associated antigen or an immunogenic fragment of a tumor- associated antigen, and (b) a polynucleotide vector encoding the same polypeptide immunogen, where the polypeptide immunogen and the polynucleotide-based vector are administered at about the same time, in different limbs of the subject, and where the subject's cancer expresses the tumor-associated antigen.
A further aspect of the present invention is a method of increasing an immune response in a subject against a polypeptide immunogen, by administering (a) a polypeptide immunogen
and (b) a polynucleotide vector encoding the same polypeptide immunogen, where the polypeptide immunogen and the polynucleotide-based vector are administered at about the same time, in different limbs of the subject, and where the immune response is increased compared to that which would be achieved by administration of the polypeptide immunogen and the polynucleotide vector at about the same time to the same limb of the subject.
A further aspect of the present invention is a composition comprising a polynucleotide vector encoding a polypeptide immunogen, for use in raising an immune response in a subject by coadministration with the same polypeptide immunogen, where the polynucleotide vector and the polypeptide immunogen are administered in different limbs of the subject at about the same time.
A further aspect of the present invention is a composition comprising a polypeptide immunogen, for use in raising an immune response in a subject by coadministration with a composition comprising a polynucleotide vector encoding the same polypeptide immunogen, where the polynucleotide vector and the polypeptide immunogen are administered in different limbs of the subject at about the same time.
A further aspect of the present invention is a use of a composition comprising a polynucleotide vector encoding a polypeptide immunogen, in the manufacture of a medicament for use in raising an immune response in a subject by coadministration with a composition comprising the same polypeptide immunogen, where the polynucleotide vector and the polypeptide immunogen are administered in different limbs of the subject at about the same time.
A further aspect of the present invention is a use of a composition comprising a polypeptide immunogen, in the manufacture of a medicament for use in raising an immune response in a subject by coadministration with a composition comprising a polynucleotide vector encoding the same polypeptide immunogen, where the polynucleotide vector and the polypeptide immunogen are administered in different limbs of the subject at about the same time.
A further aspect of the present invention is a kit comprising a polynucleotide vector encoding an immunogenic polypeptide, for use in raising an immune response in a subject when coadministered with a pharmaceutical composition comprising the same immunogenic polypeptide, where the coadministration occurs in different limbs of the subject.
A further aspect of the present invention is a kit comprising (a) a polynucleotide vector encoding an immunogenic polypeptide and (b) a composition comprising the same
immunogenic polypeptide, for use in raising an immune response in a subject when the
polynucleotide vector and the immunogenic polypeptide are coadministered to different limbs of the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 graphs the MAGEA3 specific CD8 T cell responses at week two and week four, post prime, analyzed by pentamer staining on blood samples. Each bar represents the mean (with SEM) of blood tested from each mouse in the group. The Y axis is the percentage of CD8 cells that were MAGEA3 specific. The six columns shown for week 2 correspond, from left to right, with groups 1-6 as shown in Table 1. The six columns shown for week 4 also correspond, from left to right, with groups 1-6 as shown in Table 1.
Figure 2 graphs the MAGEA3 specific CD4 and CD8 T cell responses at week four, post prime, where analysis was conducted by ICS on splenocytes. Each mouse in groups 1-4 (as set forth on Table 1) was tested. The Y-axis is percentage of IFN gamma (IFNy) positive CD4 cells (circles) or CD8 cells (squares).
Figure 3 graphs the MAGEA3 specific D8 responses (by pentamer staining of blood samples) at week two, week four, week five, week eight, week nine and week ten post prime. Induction of a strong response after the second boost is demonstrated in each tested regimen. Data was generated with pooled blood samples from each group, except in week5 and week 9 when individual samples were tested.
Figure 4 graphs MAGEA3 specific CD8 responses as detected by ICS analysis of splenocytes, at week ten (two weeks after the second boost). The data demonstrates that regimens based on contralateral co-administration of viral vectors encoding a tumor-associated antigen and a tumor-associated antigen polypeptide induced strong CD4 and CD8 responses.
DETAILED DESCRIPTION
The present invention is based on the finding that concomitant administration of a polypeptide-based immunogen and a polynucleotide-based immunogen at separate anatomic sites (e.g., in contralateral limbs) results in an increase in the CD4+ and/or CD8+ response obtained, compared to the concomitant administration of such immunogens at the same anatomic site (immune response determined, for example, using the assay techniques disclosed herein). Accordingly, concomitant administration of a polypeptide-based immunogen and a polynucleotide-based immunogen in contralateral limbs of a subject is an aspect of the present invention.
Polynucleotide-based immunogens used in the present invention include those that encode a polypeptide consisting of an amino acid sequence identical to that of the administered polypeptide immunogen, those that encode a polypeptide comprising an amino acid sequence
identical to that of the administered polypeptide immunogen, those that encode an immunogenic fragment of the administered polypeptide immunogen, or those that encode a fusion protein comprising a sequence identical to that of the administered polypeptide immunogen or of an immunogenic fragment thereof. Administration of the polynucleotide-based immunogen results in an immune response to the encoded polypeptide.
In one embodiment of the invention the polypeptide encoded by the polynucleotide- based immunogen is identical to the administered polypeptide based immunogen, i.e., has 100% sequence identity. In another embodiment of the invention, the polypeptide encoded by the polynucleotide-based immunogen is substantially the same as the administered polypeptide- based immunogen, i.e., the polypeptide encoded by the administered polynucleotide and the polypeptide-based immunogen have an overall sequence identity of at least 90%, e.g. 95% or more, e.g. 98% or more, or e.g. 99% or more over the length of the immunogenic polypeptides. As used herein, reference to administration of "the same" polypeptide as both a polypeptide- based immunogen and as a polynucleotide-based immunogen refers to administration of both the polypeptide, and a polynucleotide that encodes a polypeptide having the same amino acid sequence.
In one embodiment of the present invention, the polypeptide-based immunogen is a tumor associated antigen (TA antigen), and is administered for the treatment or prophylaxis of a cancer that expresses the TA antigen. In one embodiment, MAGE- A3 protein or polypeptide, or immunogenic fragment thereof, or a fusion protein comprising a MAGE- A3 protein or polypeptide, or immunogenic fragment thereof, is administered for the treatment or prophylaxis of a MAGE-A3 expressing tumor. Such tumors may include, e.g., melanoma, non-small cell lung cancer, liver (hepatic) cancer, and bladder cancer
In one embodiment, a human subject is treated by the concomitant administration of a MAGE- A3 polypeptide immunogen and a polynucleotide vector , such as a viral vector, that encodes a MAGE-A3 polypeptide immunogen, for the treatment or prophylaxis of a MAGE-A3 expressing tumor.
As used herein "raising an immune response" refers to the production in a subject of CD4+ cells, CD8+ cells, and/or antibodies specific to the administered polypeptide immunogen. The methods of the present invention better stimulate production of CD4+ and/or CD8+ cells and /or antibodies relative to other vaccination protocols/schedules using the same polypeptide immunogens. By 'better stimulates' is meant that the intensity and/or persistence and/or breadth of the immune response is enhanced.
As used herein, "cancer" refers to malignant neoplasms, including those arising from epithelial tissues and those arising from mesodermal tissues. As used herein a "tumor" is a mass of cancerous tissue. Anatomic site of immunization / timing of administration
Methods of immunization according to the present invention comprise the concomitant administration (co-administration) to a subject of an antigenic protein/polypeptide (a
polypeptide-based immunogenic formulation), in combination with the administration of a nucleic acid encoding an antigenic protein/polypeptide (a polynucleotide-based immunogenic formulation), where the formulations are administered at separate anatomic sites.
As used herein the term "concomitant administration" or "co-administration" means that two formulations or compositions are administered to the same subject at the same time
(simultaneously) or at about the same time. "At about the same time" encompasses sequential administration where the period between administrations is due only to the speed of the individual administering the active agents, rather than an intentional period of delay between administrations, e.g., the time period necessary for a single health care practitioner to administer a first composition according to accepted clinical practices and standards, and then administer a second composition according to accepted clinical practices and standards. In one embodiment "at about the same time" encompasses administrations within a time period of fifteen minutes or less, thirty minutes or less, one hour or less, two hours or less, six hours or less, up to about twelve hours or less. Thus concomitant administration occurs in a time period of no more than about thirty minutes, or no more than about one hour, or no more than about two hours, and does not extend beyond 12 hours.
The administered immunogenic compositions used in the present invention may include an adjuvant, as is known in the art. The health care provider may mix the adjuvant and immunogen immediately prior to administration to the subject.
In one embodiment of the invention, the polypeptide-based immunogenic composition is administered to one limb of the subject, and the polynucleotide-based immunogenic
composition is administered to a contralateral limb of the subject (e.g., right arm and left arm, or left leg and right arm).
In another embodiment of the invention, the polypeptide-based immunogenic composition is administered to one limb of the subject, and the polynucleotide-based
immunogenic composition is administered to a separate limb of the subject, including ipsilateral limbs (e.g., right arm and right leg, left arm and left leg).
As used herein, administration to 'the same anatomic site' refers to administration in the same limb, or within an anatomic area that drains to the same set of lymph nodes.
Administration may be by any suitable route as is known in the art, and includes intramuscular injection, subcutaneous injection, intradermal delivery, epidermal delivery and transdermal delivery.
Thus in one embodiment of the invention there is provided a method of raising an immune response which comprises administering (i) one or more immunogenic polypeptides, and (ii) one or more polynucleotide-based vectors (e.g., viral vectors) comprising one or more heterologous polynucleotides encoding one or more immunogenic polypeptides; wherein (i) and (ii) are administered concomitantly but at separate anatomic sites. Optionally, (i) and (ii) may be co-formulated with an adjuvant. The immunogenic polypeptide encoded by the
polynucleotide vector may consist of or comprise the same, or substantially the same, amino acid sequence as the administered immunogenic polypeptide, or consist of or comprise an immunogenic fragment of the administered polypeptide immunogen.
Typically the polypeptide-based immunogen is contained in a pharmaceutical comnposition comprising additional pharmaceutically acceptable components, e.g. excipients and carriers. Typically the polynucleotide-based immunogen is contained in a pharmaceutical comnposition comprising additional pharmaceutically acceptable components, e.g., excipients and carriers.
Methods according to the invention may involve use of more than one immunogenic polypeptide and/or more than one polynucleotide-based immunogen.
In each administration, the polypeptide-based immunogen is administered in such an amount as is capable of inducing an immune response. The immune response may be useful as as prophylactic (to prevent disease, including preventing a recurrence of disease) or as a therapeutic (to treat existing disease).
Suitably a desired immune response is achieved by a single concomitant administration (co-administration) of polypeptide-based and polynucleotide-based immunogens according to the methods of the invention (an initial administration). However the immune response may be supplemented or further enhanced by one or more subsequent administrations of said immunogens, where said subsequent administration occurs at about two weeks after the initial administration, at about four weeks after the initial administration, at about eight weeks after the initial administration, or within about one month after the initial administration, or within about two months after the initial administration, or within about six months of the initial
administration.
Polypeptide-based immunogens.
As used herein, the term "epitope" refers to an immunogenic amino acid sequence. An epitope may refer to a minimum amino acid sequence of typically 6-8 amino acids which minimum sequence is immunogenic when removed from its natural context, for example when transplanted into a heterologous polypeptide. An epitope may also refer to that portion of a protein which is immunogenic, where the polypeptide containing the epitope is referred to as the antigen (or sometimes "polypeptide antigen"). A polypeptide or antigen may contain one or more (e.g., 2 or 3 or more) distinct epitopes. The term "epitope" embraces B-cell and T-cell epitopes. The term "T-cell epitope" embraces CD4+ T-cell epitopes and CD8+ T-cell epitopes (sometimes also referred to as CTL epitopes).
The term "immunogenic polypeptide" refers to a polypeptide which is immunogenic, that is to say it is capable of eliciting an immune response in an animal, and therefore contains one or more epitopes (e.g., T-cell and/or B-cell epitopes). Immunogenic polypeptides may contain one or more epitopes, and these may be in a natural or an unnatural arrangement, including epitopes from different polypeptides, as in a fusion protein.
Immunogenic polypeptides will typically be recombinant proteins produced, e.g., by expression in a heterologous host such as a bacterial host, in yeast or in cultured mammalian cells.
Suitably the immunogenic polypeptide comprises at least one T cell epitope. Suitably the immunogenic polypeptide further comprises at least one B cell epitope.
Immunogenic polypeptides used in the present invention may be in the form of immunogenic derivatives or immunogenic fragments of immunogenic polypeptides. As used herein the term "immunogenic derivative" in relation to an immunogenic polypeptide of native origin refers to an antigen modified in a limited way relative to its native counterpart. For example the immunogenic derivative may include a point mutation which may change the properties of the protein relative to the native counterpart, e.g. by improving expression in prokaryotic systems or by removing undesirable activity, e.g. enzymatic activity. Immunogenic derivatives will however be sufficiently similar to the native antigens such that they retain their antigenic properties and remain capable of raising an immune response against the native antigen. Whether or not a given derivative raises such an immune response may be measured by a suitable immunological assay such as an ELISA (for antibody responses) or flow cytometry using suitable staining for cellular markers (for cellular responses).
As used herein, an "immunogenic fragment" of a polypeptide is a fragment which encodes at least one epitope of the polypeptide, for example a cytotoxic T lymphocyte (CTL) epitope, and is typically at least 8 amino acids in length. Fragments of at least 8, for example 8 to 10 amino acids or up to 20, 50, 60, 70, 100, 150 or 200 amino acids in length are considered to fall within the scope of the invention as long as the polypeptide demonstrates antigenicity, that is to say that the major epitopes (e.g., CTL epitopes) are retained by the polypeptide fragment.
Polynucleotide delivery systems:
As used herein, polynucleotide-based immunogens are those delivered by a nucleic acid encoding a polypeptide immunogen. Polynucleotide-based immunogens comprise expression vectors that enter cells and express the polypeptide immunogen. The terms refers to both nucleic acids administered as a purified form, e.g., an expression plasmid, as well as nucleic acids that are administered as a virus (within a viral capsid). The nucleic acid encodes immunogenic epitopes from an antigen of interest that induces humoral and/or cellular immune responses. The nucleic acid component can be any nucleic acid, including DNA or RNA. For example, in some embodiments, the nucleic acid component can be a viral RNA or a messenger RNA.
Various methods of administering a polynucleotide encoding a polypeptide immunogen are known in the art, including nucleic acid expression systems such as plasmid DNA, bacterial and viral expression systems. Where the subject to be immunized is a mammal, the
polynucleotide sequence encoding the immunogen may be codon optimized for expression in mammalian cells. Desirably the codon usage pattern of the polynucleotide sequence is typical of highly expressed human genes. The principle of such codon optimization is described, for example, in WO 05/025614. When several antigens or epitopes are provided as a fusion protein, such fusion would be encoded by a polynucleotide under the control of a single promoter.
Administration of naked DNA by injection into mouse muscle is outlined by Vical in International Patent Application W090/11092. Johnston et al WO 91/07487 describe methods of transferring a gene to vertebrate cells via microprojectiles coated with a polynucleotide encoding a gene of interest, and accelerating the microparticles such that the microparticles penetrate the target cell. DNA vaccines may comprise a bacterial plasmid vector into which is inserted a viral promoter, a sequence encoding the antigenic peptide to be delivered, and polyadenylation/transcriptional termination sequences. The sequence may encode a full protein, a fusion protein comprising different antigens, or simply an antigenic peptide sequence against which an immune response is desired. Following administration the plasmid is taken up by cells
of the host (e.g., a vaccinated subject) where the encoded peptide is produced. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the host
(such as a suitable promoter and terminating signal).
Alternatively, the system for expressing the immunogen may be a recombinant live microorganism, such as a virus or bacterium into which the sequence expressing the
immunogen of interest is inserted. Inoculation and in vivo infection of a subject with such a live vector leads to in vivo expression of the antigen and induction of an immune response. Thus, viral vectors used in the present invention comprise one or more heterologous polynucleotides which encode one or more immunogenic polypeptides.
The viral vector may be any suitable viral vector. Viruses useful for expressing an immunogen include poxviruses (e.g; vaccinia, fowlpox, canarypox, modified poxviruses e.g.
Modified Virus Ankara (MVA)), alphaviruses (Sindbis virus, Semliki Forest Virus,
Venezuelian Equine Encephalitis Virus), flaviviruses (yellow fever virus, Dengue virus,
Japanese encephalitis virus), adenoviruses, adeno-associated virus, picomaviruses (poliovirus, rhinovirus), herpesviruses (varicella zoster virus, etc). These viruses can be attenuated in various ways in order to obtain live vaccines.
The adenoviruses are a large family of double-stranded DNA viruses. Wold &
Tollefson, Adenovirus methods and protocols: Adenoviruses, Ad Vectors, Quantitation, and
Animal models; 2nd edition, Humana Press: 2007.
Virus types include: dsDNA viruses (e.g. Adenoviruses, Herpesviruses, Poxviruses); ssDNA viruses (+) sense DNA (e.g. Parvoviruses); dsRNA viruses (e.g. Reoviruses);
(+)ssRNA viruses (+) sense RNA (e.g. Picomaviruses, Togaviruses); (-)ssRNA viruses (-) sense
RNA (e.g. Orthomyxoviruses, Rhabdoviruses); ssRNA-RT viruses (+) sense RNA with DNA intermediate in life-cycle (e.g. Retroviruses); dsDNA-RT viruses (e.g. Hepadnaviruses).
DNA virus types include: Adenoviridae; Papillomaviridae; Parvoviridae; Herpesviridae e.g. Herpes simplex virus, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus;
Poxyiridae e.g. Smallpox virus, vaccinia virus; Hepadnaviridae e.g. Hepatitis B virus;
Polyomaviridae e.g. Polyoma virus, JC virus (progressive multifocal leucoencephalopathy);
Circoviridae e.g. Transfusion Transmitted Virus. RNA virus types include Reoviridae e.g.
Reovirus, Rotavirus; Picornaviridae e.g. Enterovirus, Rhinovirus, Hepatovirus, Cardiovirus,
Aphthovirus, Poliovirus, Parechovirus, Erbovirus, Kobuvirus, Teschovirus, Coxsackie;
Caliciviridae e.g. Norwalk virus, Hepatitis E; Togaviridae e.g. Rubella virus; Arenaviridae e.g.
Lymphocytic choriomeningitis virus; Flaviviridae e.g. Dengue virus, Hepatitis C virus, Yellow fever virus; Orthomyxoviridae e.g. Influenzavirus A, Influenzavirus B, Influenzavirus C,
Isavirus, Thogotovirus; Paramyxoviridae e.g. Measles virus, Mumps virus, Respiratory syncytial virus; Bunyaviridae e.g. California encephalitis virus, Hantavirus; Rhabdoviridae e.g. Rabies virus; Filoviridae e.g. Ebola virus, Marburg virus; Coronaviridae e.g. Corona virus; Astroviridae e.g. Astrovirus; Bornaviridae e.g. Borna disease virus. RT virus types include Metaviridae; Pseudoviridae; Retroviridae— e.g. HIV; Hepadnaviridae— e.g. Hepatitis B virus; Caulimoviridae— e.g. Cauliflower mosaic virus.
In one aspect of the invention the vector is the measles virus. Measles virus (MV) belongs to the genus Morbillivirus in the family Paramyxoviridae. Measles viral vectors are disclosed in, by way of example, WO2008/078198, WO 2006/136697, WO2004/001051 and WO2004/000876, Combredet et al., Journal of Virology 77 (21): 11546-11554 (2003).
In one aspect of the invention the vector is selected from the adenoviral vectors described in PCT/EP2010 000616 (published as WO 2010/086189 and US 2012-0027788 ) and PCT/EP2009/000672 (published as WO 2010/085984). In one aspect of the invention the vector is selected from the replication defective viral vectors ChAd83 and PanAd3.
The nucleic acid expression system is administered in sufficient amounts to transduce the target cells and provide sufficient levels of gene transfer and expression and to permit specific immune responses to the administered immunogen to develop. Such dosages will vary depending upon the immunogen administered, the condition being treated, the subject being treated, and other factors, and can be determined by those skilled in the medical arts.
The polynucleotide or polynucleotides encoding immunogenic polypeptides to be expressed may be inserted into any suitable region of the viral vector, for example into a deleted region. Although two or more polynucleotides encoding immunogenic polypeptides may be linked as a fusion, the resulting protein may be expressed as a fusion protein, or it may be expressed as separate protein products, or it may be expressed as a fusion protein and then subsequently broken down into smaller subunits.
In one aspect the viral vector is suitably replication competent in the host organism to which it is to be delivered. In another aspect the viral vector is attenuated or replication defective (unable to replicate in mammalian hosts, e.g., Fowlpoxvirus (FPV) and modified vaccinia virus Ankara strain (MVA)). Such constructs are replication deficient and non- integrating, and deliver the immunogen (antigen) into the antigen-processing pathways of transfected or infected cells.
The viral vector of the invention may be replication defective. This means that it has a reduced ability to replicate in non-complementing cells, compared to the wild type virus. This may be brought about by mutating the virus e.g. by deleting a gene involved in replication.
In a further aspect the viral vector is not affected by, or only minimally affected by the presence of an adjuvant. In one aspect any reduction in viral titer caused by the adjuvant is no more than 50%, such as no more than 40%, 30%, 20%, 15%, 10%, 5% and in a further aspect there is no reduction in titer at all.
ASCI and therapeutic cancer vaccines.
In one embodiment, the methods of the present invention may be used to deliver therapeutic anti-cancer vaccines.
Antigen-based immunotherapy to treat cancer utilizes tumor-associated antigens to elicit specific host immune responses against the antigen, in order to train the patient's immune system to eliminate tumor cells. As used herein, a tumor-associated antigen is a protein frequently expressed by a tumor type and capable of inducing an immune response in the subject. Certain tumor-associated antigens are expressed selectively or preferentially on cancer cells. Tumor antigens can be classified into 5 groups: (1) cancer-testis antigens that are expressed in a range of different tumor types but not in normal tissues except testis (e.g., MAGE and NY-ESO-1), (2) melanocyte differentiation antigens expressed in melanoma and normal melanocytes (e.g. gplOO and tyrosinase); (3) antigens encoded by mutated normal gene (e.g. p53 and ras); (4) self-antigens overexpressed in malignant tissues (e.g. HER-2/ neu); and (5) antigens derived from oncogenic viruses (e.g. HPV and EBV).
Multiple tumor-associated antigens have been identified, including the cancer-testis antigens of the B AGE, GAGE, MAGE, NY-ESO- 1 , PRAME and S SX families; differentiation antigens (including GP100, Mel an- A/Mart- 1, PSA, CEA, and Mammaglobin-A); antigens overexpressed in tumor tissues (including p53, her2/neu, survivin, WT1); tumor-associated carbohydrate antigens (including Muc-1). Urban and Schreiber, Annu Rev Immunol 10:617-44 (1992); Van den Eynde et al., Curr Opin Immunol 9:684-93 (1997); Buonaguro et al., Clin Vacc Immunology 18(l):23-34 (2011). As is known in the art, tumor cells from a single tumor, or within a single subject, may be heterogeneous, that is, with some tumor cells expressing the tumor-associated antigen while other cells do not. Such tumors are, for purposes of the present invention, considered as tumor antigen-expressing tumors.
These specific tumor-associated antigens are referred to by way of example, and are not intended to be limiting upon the scope of the present invention
MAGE-A3 is a member of the MAGE family. MAGE-A3 antigenic polypeptides and fusion proteins are in clinical trials as anti-cancer vaccine antigens. See, e.g., Vansteenkiste et al., J. Clin. Oncol 31 :2396-403 (2013); Kruit et al., J. Clin Oncol. 31 :413-20 (2013); Kruit et al., Int. J. Cancer 117:596-604 (2005); Marchand et al., Eur. J. Cancer 39:70-77 (2003);
Atanackovic et al., Proc. Natl. Acad. Sci. 105: 16-50-1655 (2008); Vantomme et al., J.
Immunother 27: 124-135 (2004); Atanackovic et al., J. Immunol. 172:3289-96 (2004).
The human MAGE-A3 sequence is known in the art, see e.g., GenBank Accession CAG46573, NCBI Reference Sequence NP_005353.1 :
MPLEQRSQHC KPEEGLEARG EALGLVGAQA PATEEQEAAS SSSTLVEVTL GEVPAAESPD PPQSPQGASS LPTTMNYPLW SQSYEDSSNQ EEEGPSTFPD LESEFQAALS RKVAELVHFL LLKYRAREPV TKAEMLGSVV GNWQYFFPVI FSKASSSLQL VFGIELMEVD PIGHLYI FAT CLGLSYDGLL GDNQIMPKAG LLIIVLAIIA REGDCAPEEK IWEELSVLEV FEGREDSILG DPKKLLTQHF VQENYLEYRQ VPGSDPACYE FLWGPRALVE TSYVKVLHHM VKISGGPHIS
YPPLHEWVLR EGEE -314 (SEQ ID NO : 1 )
As used herein, recMAGE-A3 polypeptide immunogen refers to a specific 450-amino acid recombinant protein having SEQ ID NO:2. recMAGE-A3 contains a Protein D sequence (amino acids 1-127 of SEQ ID NO:2), inserted Met- Asp (amino acids 128-129 of SEQ ID
NO:2), a fragment of MAGE- A3 (amino acids 3-314 of MAGE- A3, at amino acids 130- 441 of SEQ ID No: 2), inserted Gly-Gly (amino acids 442-443 of SEQ ID NO:2)and a seven histidine tail (amino acids 444-450 of SEQ ID NO:2). See e.g., WO99/40188; US Patent No. 8,097,257. MDPKTLALSL LAAGVLAGCS SHSSNMANTQ MKSDKI I IAH RGASGYLPEH - 50
TLESKALAFA QQADYLEQDL AMTKDGRLW IHDHFLDGLT DVAKKFPHRH -100
RKDGRYYVID FTLKEIQSLE MTENFETMDL EQRSQHCKPE EGLEARGEAL -150
GLVGAQAPAT EEQEAASSSS TLVEVTLGEV PAAESPDPPQ SPQGASSLPT -200
TMNYPLWSQS YEDSSNQEEE GPSTFPDLES EFQAALSRKV AELVHFLLLK -250 YRAREPVTKA EMLGSWGNW QYFFPVIFSK ASSSLQLVFG IELMEVDPIG -300
HLYIFATCLG LSYDGLLGDN QIMPKAGLLI IVLAI IAREG DCAPEEKIWE -350
ELSVLEVFEG REDSILGDPK KLLTQHFVQE NYLEYRQVPG SDPACYEFLW -400
GPRALVETSY VKVLHHMVKI SGGPHISYPP LHEWVLREGE EGGHHHHHHH*-450
(SEQ ID NO:2)
Although MAGE- A3 -specific antibodies may not directly be involved in removing tumor cells, they may contribute to CD8+ T-cell cytotoxic activity because antibody-mediated opsonization of MAGE- A3 has been found to promote cross-presentation to naive T cells. Moeller et al., Cancer Immunol. Immunother. 61 :2079 (2012).
Accordingly, in one embodiment of the present invention, where vaccination is intended as a therapeutic treatment for the presence of a tumor that expresses MAGE- A3, or as a prophylactic treatment for a subject who has previously undergone treatment for or been diagnosed with a MAGE- A3 expressing tumor, a MAGE-A3 immunogenic polypeptide and a polynucleotide-based vector encoding a MAGE-A3 immunogenic polypeptide are
concomitantly administered to the subject at different anatomic sites. Such administration (the
vaccination) may occur after surgical, chemotherapeutic, radiologic, or other treatment of the tumor, including in cases where, subsequent to such treatment, there is no evidence of disease.
An adjuvanted recombinant MAGE- A3 fusion polypeptide of SEQ ID NO:2 has been used in clinical trials for the therapy of cancer, including where the adjuvant was AS02B or AS 15, as described herein. Clinical trials of adjuvanted, recombinant MAGE- A3 fusion polypeptide have included subjects with Non-small cell lung cancer (NSCLC), melanoma, and bladder cancer. See, e.g., Peled et al., Immunotherapy 1(1): 19-25 (209).
Accordingly, one embodiment of the present invention is the co-administration of an immunogenic polypeptide having SEQ ID NO:2 and a polynucleotide-based immunogen (such as a viral vector) encoding a full length MAGE A3 polypeptide (such as a polypeptide of SEQ ID NCv l).
Adjuvants
An "adjuvant" as used herein refers to a composition that enhances the immune response to an immunogen. Adjuvants are described in general, e.g. in Vaccine Design—the Subunit and Adjuvant Approach, Powell and Newman, Plenum Press, New York, 1995.
An immune response is generated to an antigen through the interaction of the antigen with the cells of the immune system. The resultant immune response may be broadly classified into two categories: the humoral and cell mediated immune responses (traditionally
characterised by antibody and cellular effector mechanisms of protection, respectively). These categories of response have been termed Thl-type responses (cell-mediated response), and Th2- type immune responses (humoral response).
Extreme Thl-type immune responses may be characterized by the generation of antigen specific, haplotype restricted cytotoxic T lymphocytes, and natural killer cell responses. In mice, Thl-type responses are often characterised by the generation of antibodies of the IgG2a subtype, whilst in the human these correspond to IgGl type antibodies. Th2-type immune responses are characterised by the generation of a broad range of immunoglobulin isotypes including in mice IgGl, IgA, and IgM. However, the distinction of Thl and Th2-type immune responses is not absolute. In reality an individual will support an immune response which is described as being predominantly Thl or predominantly Th2.
It is known that certain vaccine adjuvants are suited to the stimulation of either Thl or
Th2-type cytokine responses. Traditionally the best indicators of the Thl :Th2 balance of the immune response after a vaccination or infection includes direct measurement of the production of Thl or Th2 cytokines by T lymphocytes in vitro after re-stimulation with antigen, and/or the measurement of the IgGl :IgG2a ratio of antigen specific antibody responses. The nature of the
T-cell response to a vaccine immunogen can be influenced by the composition of the adjuvant used in the vaccine. For instance, adjuvants containing MPL & QS21 have been shown to activate Thl CD4+ T cells to secrete IFN-gamma (Stewart et al. Vaccine. 2006, 24 (42- 43):6483-92).
Thus, a Thl-type adjuvant is one which stimulates isolated T-cell populations to produce high levels of Thl-type cytokines when re-stimulated with antigen in vitro, and induces antigen specific immunoglobulin responses associated with Thl-type isotype.
One suitable adjuvant is monophosphoryl lipid A (MPL), in particular 3-de-O-acylated monophosphoryl lipid A (3D-MPL). Chemically it is often supplied as a mixture of 3-de-O- acylated monophosphoryl lipid A with either 4, 5, or 6 acylated chains. It can be purified and prepared by the methods taught in GB 2122204B, which reference also discloses the preparation of diphosphoryl lipid A, and 3-O-deacylated variants thereof. Other purified and synthetic lipopolysaccharides have been described (U.S. Pat. No. 6,005,099 and EP 0 729 473 B l;
Hilgers et al., 1986, Int.Arch. Allergy. Immunol., 79(4):392-6; Hilgers et al., 1987, Immunology, 60(1): 141-6; and EP 0 549 074 Bll).
Saponins are also known Thl immunostimulants. Lacaille-Dubois, M and Wagner H, A review of the biological and pharmacological activities of saponins. Phytomedicine vol 2 pp 363-386 (1996). For example, the saponin Quil A (derived from the bark of the South American tree Quillaja Saponaria Molina), and fractions thereof, are described in U.S. Pat. No. 5,057,540 and Kensil, Crit. Rev. Ther. Drug Carrier Syst, 1996, 12: 1-55; and EP 0 362 279 B l . Purified fractions of Quil A are also known as immunostimulants, such as QS21 and QS17; methods of their production is disclosed in U.S. Pat. No. 5,057,540 and EP 0 362 279 B l . Also described in these references is QS7 (a non-haemolytic fraction of Quil-A). Use of QS21 is further described in Kensil et al. (1991, J. Immunology, 146: 431-437). Combinations of QS21 and polysorbate or cyclodextrin are also known (WO 99/10008). Particulate adjuvant systems comprising fractions of QuilA, such as QS21 and QS7 are described in WO 96/33739 and WO 96/11711.
Another immunostimulant is an immunostimulatory oligonucleotide containing unmethylated CpG dinucleotides ("CpG") (Krieg, Nature 374:546 (1995)). CpG is an abbreviation for cytosine-guanosine dinucleotide motifs present in DNA. CpG is known as an adjuvant when administered by both systemic and mucosal routes (WO 96/02555, EP 468520, Davis et al, J.Immunol, 1998, 160:870-876; McCluskie and Davis, J.Immunol., 1998, 161 :4463- 6). CpG, when formulated into vaccines, may be administered in free solution together with free antigen (WO 96/02555; McCluskie and Davis, supra) or covalently conjugated to an antigen
(WO 98/16247), or formulated with a carrier such as aluminium hydroxide (Davis et al, supra ; Brazolot-Millan et al., Proc. Natl. Acad. Sci., USA, 1998, 95: 15553-8).
Immunostimulants such as those described above may be formulated together with carriers, such as liposomes, oil in water emulsions, and or metallic salts (including aluminum salts such as aluminum hydroxide). For example, 3D-MPL may be formulated with aluminum hydroxide (EP 0 689 454) or oil in water emulsions (WO 95/17210); QS21 may be formulated with cholesterol containing liposomes (WO 96/33739), oil in water emulsions (WO 95/17210) or alum (WO 98/15287); CpG may be formulated with alum (Davis et al, supra ; Brazolot- Millan, supra) or with other cationic carriers.
Combinations of immunostimulants may be utilized in the present invention, in particular a combination of a monophosphoryl lipid A and a saponin derivative (see, e.g., WO 94/00153; WO 95/17210; WO 96/33739; WO 98/56414; WO 99/12565; WO 99/11241), more particularly the combination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a composition where the QS21 is quenched in cholesterol-containing liposomes (DQ) as disclosed in WO 96/33739. Alternatively, a combination of CpG plus a saponin such as QS21 is an adjuvant suitable for use in the present invention. A potent adjuvant formulation involving QS21, 3D-MPL & tocopherol in an oil in water emulsion is described in WO 95/17210 and is another formulation for use in the present invention. Saponin adjuvants may be formulated in a liposome and combined with an immunostimulatory oligonucleotide. Thus, suitable adjuvant systems include, for example, a combination of monophosphoryl lipid A, preferably 3D-MPL, together with an aluminium salt (e.g. as described in WO00/23105). A further exemplary adjuvant comprises comprises QS21 and/or MPL and/or CpG. QS21 may be quenched in cholesterol-containing liposomes as disclosed in WO 96/33739.
Kruit et al. (J. Clin. Oncology 19: 2413-2420 (2013)) reports on the comparison of the immunostimulant systems AS02B and AS 15 for use with recombinant MAGE- A3 protein
(recMAGE-A3) for active immunization. Immunostimulant AS02B is a combination of QS21 saponin and monophosphoryl lipid A (a TLR-4 agonist) in an oil/water emulsion.
Immunostimulant AS 15 is a combination of QS21, monophosphoryl lipid A, and CpG7909 (a TLR-9 agonist), in a liposomal formulation. Kruit et al. (2013) concluded that clinical activity seemed to be higher when recMAGE- A3 was used with AS 15.
Other suitable adjuvants include alkyl Glucosaminide phosphates (AGPs) such as those disclosed in WO9850399 or U.S. Pat. No. 6,303,347 (processes for preparation of AGPs are also disclosed), or pharmaceutically acceptable salts of AGPs as disclosed in U.S. Pat. No.
6,764,840. Some AGPs are TLR4 agonists, and some are TLR4 antagonists. Both are thought to be useful as adjuvants.
Suitably the adjuvant component does not contain any virus. Thus suitably,
compositions for use according to the invention do not contain any virus other than the one or more more viral vectors comprising one or more heterologous polynucleotides encoding one or more immunogenic polypeptides.
The amount of adjuvant will depend on the nature of the adjuvant and the immunogenic polypeptide, the condition being treated and the age, weight and health of the subject. Compositions
Compositions administered as part of the methods described herein may be formulated for pharmaceutical administration. Carriers used in pharmaceutical compositions will vary, as is known in the art, depending upon the route and mode of administration. Subjects
The present methods of immunization (vaccination) are suitable for use in mammalian subjects, including humans, non-human primates, small mammals including rodents (mice and rats, e.g.) and rabbits, and domesticated animals such as cattle, horses, sheep, dogs and cats.
The immunogenic compositions are co-administered to subjects in amount(s) sufficient to elicit an immune response , e.g., a CD8, CD4, and/or antibody response to the antigen of interest to which the nucleic acid/protein components are directed. In one embodiment the amount of immunogenic compositions are sufficient to induce a therapeutic immune response, e.g., a response that at least partially arrests or slows symptoms, progression, and/or complications of a disease, e.g. cancer. An amount adequate to accomplish this is defined as "therapeutically effective dose."
In one embodiment the amount of immunogenic compositions are sufficient to induce a prophylactic immune response, e.g., a response that at least partially reduces the incidence or prevalence of disease. An amount adequate to accomplish this is defined as a "prophylactically effective dose."
Amounts effective to achieve therapeutic or prophylactic immune responses will depend on, e.g., the particular composition of the regimen administered, the manner of administration, the stage and severity of the disease, the general state of health of the patient, and the judgment of the prescribing health care provider.
Assessment of Immunogenic Response
To assess an individual's immune system during and after administration of an immunogen, co-administration of immunogens, or sequential administration of immunogens, various parameters can be measured as are known in the art. Measurements to evaluate vaccine response include but are not limited to: antibody measurements in the plasma, serum, saliva, or other body fluids; analysis of in vitro cell proliferation in response to a specific antigen, indicating the function of CD4' cells; analysis of cytokine production of lymphocytes after stimulation with the specific antigen or with pools of peptides of the specific antigen; and analysis ofneutralizing activity found in the serum or plasma using virus inhibition as says well known in the art. Such assays are well known in the art.
Other measurements of immune response include assessing CD8+ responses, for example, using tetramer staining of fresh or cultured PBMC (see, e.g., Altman, et al. , Proc. Natl. Acad. Sci. USA 90: 10330, 1993; Altman, et al. , Science 274:94, 1996), or 7-interferon release assays such as ELISPOT assays (see, e.g., Lalvani, et al. , J. Exp. Med. 186:859, 1997; Dunbar, et al. , Curr. Biol. 8:413, 1998; Murali-Krishna, et al. , Immunity 8: 177, 1998), or by using functional cytotoxicity assays.
In other embodiments, the antibody response is measured.
Kits
A further aspect of the present invention are kits comprising the polynucleotide and polypeptide components to be administered in accordance with the methods described herein. Such a kit can comprise for example, a container that includes one or more of the
polynucleotide vectors contained in a vessel and a separate container containing the polypeptide form of the antigen. In one embodiment, polypeptide is formulated with an adjuvant; in another embodiment the adjuvant is contained in a separate container and is admixed with the polypeptide immunogen prior to administration. Thus, a kit of the invention can comprise a polypeptide form of the antigen separate from the polynucleotide form of the antigen, and may comprise a separate adjuvant. The kit may also include additional components, e.g., for mixing with one or both of the compositions before administration, such as diluents, carriers, and the like.
Terms & Miscellaneous
Unless otherwise explained, 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 belongs.
The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "plurality" refers to two or more. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Additionally, numerical limitations given with respect to concentrations or levels of a substance, such as an antigen, are intended to be approximate. Thus, where a concentration is indicated to be at least (for example) 200 pg, it is intended that the concentration be understood to be at least approximately (or "about" or "~") 200 pg.
The terms "peptide, ""polypeptide, " and "protein" are used interchangeably herein to refer to at least two amino acids or amino acid analogs that are covalently linked by a peptide bond or an analog of a peptide bond. The amino acids of the peptide may be L-amino acids or D-amino acids. A peptide, polypeptide or protein may be synthetic, recombinant or naturally occurring. A synthetic peptide is a peptide produced by artificial means in vitro.
As used herein, the terms "identical" in the context of two or more nucleic acids or two or more polypeptide sequences, refers to two or more sequences that have the same nucleotide or amino acids sequence, respectively. As used herein, the terms "similar" or "substantially similar" or "substantially the same" refer to sequences having a specified percentage of amino acid residues or nucleotide residues that are the same (ie., about 70% identity, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region of an antigen of interest or a nucleic acid encoding an antigen of interest when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms, or by manual alignment and visual inspection (see, e.g., NCBI web site or the like). The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. A preferred example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST algorithms, which are described in Altschul et al. , Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al. , J.Mol. Biol. 215:403-410(1990),respectively. BLAST software is publicly available through the National Center for Biotechnology Information, e.g., on the worldwide web..
The terms "enhanced immune response" or "increased immune response" as used herein refers to an immune response that includes increases in the level of immune cell activation,
and/or an increase in the duration of the response, and/or improved immunological memory, and/or an improvement in the kinetics of the immune response. Such increases can be demonstrated by either a numerical increase, e.g., an increased in levels of antibody in a particular time frame, as assessed in an assay to measure the response assay, or by prolonged longevity of the response.
Antigen is used herein as synonymous with immunogen, and antigenic is used herein as synonymous with immunogenic.
As used herein, "homolateral" or "ipsilateral" administration refers to administration in the limbs on one side of a subject (i.e., administrations in the left arm and left leg of a subject are considered as ipsilateral administration). As used herein, "contralateral administration" means administration in limbs on the opposite sides of a subject (e.g., separate administrations in the left arm and the right arm of a subject are considered to be contralateral administration, as are separate administrations in the left leg and right leg of a subject). Administrations to the same limb of a subject are considered to be at the same anatomic site. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." Thus, unless the context requires otherwise, the word "comprises," and variations such as "comprise" and "comprising" will be understood to imply the inclusion of a stated compound or composition (e.g., nucleic acid, polypeptide, antigen) or step, or group of compounds or steps, but not to the exclusion of any other compounds, composition, steps, or groups thereof. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."
By "immunogen-specific CD4+ T-cells and/or CD8+ T-cells and/or antibodies" is meant CD4+ T-cells and/or CD8+ T-cells and/or antibodies which specifically recognise a specific immunogen or part thereof (e.g., an epitope thereof). By "specifically recognise" is meant that said CD4+ T-cell and/or CD8+ T-cell recognition is immunospecific rather than a non-specific.
The application of which this description and claims forms part may be used as a basis for priority in respect of any subsequent application. The claims of such subsequent application may be directed to any feature or combination of features described herein. They may take the form of product, composition, process, or use claims and may include, by way of example and without limitation, the claims appended herein.
All references referred to in this application, including patent and patent applications, are incorporated herein by reference to the fullest extent possible. The following examples and data illustrate the invention, but are not limiting upon the invention.
EXAMPLES
As used in the Examples herein, "recMAGE-A3 AS-15 adjuvanted protein" refers to a composition comprising the recMAGE-A3 polypeptide of SEQ ID NO:2 and AS 15 adjuvant. AS 15 (Adjuvant System 15) contains MPL, QS21, and CpG in a liposomal formulation.
PanAd3-MAGEA3, as used in the Examples herein, refers to the chimpanzee viral vector PanAd3 (as described in PCT/EP/2010/000616 (published as WO 2010/086189) see also EP10702615 and US 13/147, 193) expressing a full-length MAGE-A3 protein of SEQ ID NO: l .
Adenovirus ChAd83-MAGEA3 as used in the Examples herein, refers to the
chimpanzee viral vector ChAd83 (as described in PCT/EP/2010/000616 (published as WO 2010/086189) see also EP10702615 and US 13/147,193) expressing a full-length MAGE-A3 protein of SEQ ID NO: 1.
MVA-MAGEA3 as used in the Examples herein, refers to a Modified Vaccinia Ankara viral vector expressing a full-length MAGE- A3 protein of SEQ ID NO: 1. MVA is a highly attenuated strain of vaccinia virus; see, e.g., Mayr et al., Zentralbl Bakteriol. B. 167(5-6):375-90 (1978) and Mayr et al., Infection 3 :6-14 (1975). Example 1: Materials & Methods for Comparison of Co-administered schedules in Mice
Female CB6F1 mice (six weeks old) were purchased from Harlan Laboratories.
Experimental groups consisted of five mice each. Mice were vaccinated intramuscularly (in the quadriceps) with PanAd3-MAGEA3 at the final dose of 5xl06vp (vp = viral protein) and with recMAGEA3 AS 15-adjuvanted protein corresponding to 1 μg according to the following schedules (see also Tablel).
Group 1 and 2 received, respectively, PanAd3-MAGEA3 vector given in one site (25 μΐ corresponding to 5xl06vp) or given contralaterally (25 μΐ per injection corresponding to 2.5 xl06vp each injection for a total dose of 5xl06vp).
Groups 3 and 4 received the co-administration of Pan Ad3 -MAGE A3 and recMAGEA3
AS 15-adjuvanted protein (25 μΐ each vaccine) given in contralateral sites (Group 3) or in one site (Group 4).
Group 5 received 50μ1 of co-formulated Pan Ad3 -MAGE A3 and recMAGEA3 AS 15 adjuvanted protein in one site. Co-formulation for this group was obtained by mixing 25 μΐ of Pan Ad3 -MAGE A3 (5xl06vp) and 25 μΐ of recMAGEA3 AS 15-adjuvanted protein (^g).
Group 6 was injected with only 25 μΐ recMAGEA3 AS 15 -adjuvanted protein (Ι μ ).
As used herein, "one site" refers to injection in the same limb. The vaccination schedule was: intramuscular (EVI) administration ΓΜ at week zero(0), with analysis at two (2) and four (4) weeks post administration.
Thus the experimental groups were (see Table 1 also):
Group 1 : PanAd3 -MAGE (at single injection site)
Group 2: PanAd3-MAGE (at two contralateral injection sites)
Group 3 : Pan Ad3 -MAGE + recMAGE-A3 AS- 15 adjuvanted protein (Pan Ad3 -Mage
injected at one site; recMAGE-A3 AS-15 adjuvanted protein injected at second contralateral site):
Group 4: PanAd3-MAGE + recMAGE-A3 AS-15 adjuvanted protein (PanAd3-Mage and recMAGE-A3 AS-15 adjuvanted protein injected separately, at same site)
Group 5 : PanAd3-MAGE + recMAGE-A3 AS-15 adjuvanted protein (PanAd3-Mage and recMAGE-A3 AS-15 adjuvanted protein co-formulated into a single injection)
Group 6: recMAGE-A3 AS-15 adjuvanted protein (one site)
Table 1
Group Vaccine Site Volume per Dose Ad per Dose ASCI Total Dose
injection injection per injection (Ad/ASCI)
1 PanAd3-MAGE Left 25ul 5X106vp - 5X106vp
(one site)
2 PanAd3-MAGE Left & 25ul each 2.5X106vp - 5X106vp
(contralateral) Right
3 PanAd3-MAGE Left & 25ul each 5X106vp lug 5X106vp/
+ASCI* Right lug
(contralateral)
4 PanAd3-MAGE Left 25ul each 5X106vp l ug 5X106vp/
+ASCI* lug
(one site)
5 PanAd3-MAGE Left 25ul each, co- 5X106vp lug 5X106vp/
+ASCI* formulated to lug
(one site, 50 ul
co-formulated)
6 ASCI* Right 25ul - lug l ug
(one site, 25 ul)
* ASCI = Antigen Specific Cancer Immunotherapy and in the Tables herein, refers to recMAGE-A3 AS- 15 adjuvanted protein.
Example 2: Materials & Methods for MAGEA3 prime/boost with viral vectors and ASCI
The immunogenicity of sequential (prime-boost) administrations of polynucleotide vectors and polypeptide immunogens was evaluated, using different administration schedules.
Female CB6F1 mice (six weeks old) were purchased from Harlan Laboratories;
experimental groups consisted of five mice each. Mice were all primed intramuscularly (in the quadriceps) with Pan Ad3 -MAGE A3 alone in a volume of 50μ1 corresponding to 5xl06 vp (Groups 5-6-7), or with using concomitant contralateral administration of Pan Ad3 -MAGE A3 and 50μ1 recMAGEA3 AS 15 -adjuvanted protein corresponding to ^g (Groups 1-2-3). Control group (Group 4) was primed with 50μ1 of recMAGEA3 AS 15 -adjuvanted protein corresponding to ^g.
Animals were then all boosted at week 4 and week 8 according to the scheme of Table2. Administration of recMAGEA3 AS 15 -adjuvanted protein was performed alternating the sites of injection for all the tested groups receiving multiple administration of the protein. Boosting vectors Pan Ad3 -M AGEA3 , ChAd83 -MAGEA3 , MVA-MAGEA3 were all injected in a volume of 50μ1 at dose of 5xl06 vp (PanAd3), 107 vp (ChAd83) and 107 pfu (MVA) alone or in combination (contralateral administration) with 50μ1 recMAGEA3 AS 15 -adjuvanted protein corresponding to ^g (Table2).
Seven groups of CB6F1 mice (five mice in each group) were used. Each group was vaccinated three times: at Week Zero [prime], Week Four [second immunization or first boost] and Week 8 [third immunization or second boost].
Analysis was as follows: whole blood MAGEA3 pentamer staining at week two, week four (pre-boost), week five, week 8, week 9 and week 10. Also at week ten, IFNy-ELISpot was conducted with CD 8 MAGEA3 immunodominant peptide (SEQ ID NO:4 SYVKVLHHM) and a pool of MAGE-A3 overlapping 15 mer peptides (splenocytes). Also at week 10, IFNy-ICS with a pool of MAGE- A3 overlapping 15mer peptides (splenocytes).
TABLE 2
Group Week O Week 4 Week 8
1st Immunization 2nd Immunization 3rd Immunization
(first boost) (second boost)
1 Pan Ad3 -MAGE ASCI* PanAd3-MAGE + ASCI*
+ ASCI*
2 Pan Ad3 -MAGE ASCI* ChAd83-MAGE + ASCI*
+ ASCI*
3 Pan Ad3 -MAGE ASCI* MVA-MAGE + ASCI*
+ ASCI*
4 ASCI* ASCI* ASCI*
5 Pan Ad3 -MAGE ChAd83-MAGEA3 ASCI*
6 Pan Ad3 -MAGE MVA-MAGEA3 ASCI*
7 Pan Ad3 -MAGE Pan Ad3 -MAGE ASCI*
*concomitant contralateral administration of vector and ASCI
ASCI = Antigen Specific Cancer Immunotherapy and, in the Tables herein, refers to recMAGE- A3 AS- 15 adjuvanted protein.
Example 3: Analysis Materials and Methods
IFNy ELISpot: IFN-y-producing T cells were evaluated by the following ELISPOT assay, in Experiment 1 at four weeks post vaccination, and in Experiment 2 at week 10 (two weeks post second boost).
MSIP S4510 plates (Millipore) were coated with lC^g/ml of anti-mouse (U-CyTech Utrecht, The Netherlands) overnight at 4°C. After washing and blocking, mouse splenocytes were plated in duplicate at two different densities (2 x 105 and 4 x 105 cells/well) and stimulated overnight with CD8 MAGEA3 immunodominant peptide or with a pool of MAGE- A3 overlapping 15mer peptides at final concentration of ^g/ml each peptide. The peptide diluent DMSO (Sigma- Aldrich, Milan, Italy) and the Concanavalin A (ConA) polypeptide (Sigma- Aldrich, Milan, Italy) were used respectively as negative and positive controls. Plates were developed by subsequent incubations with biotinylated anti-mouse IFNy antibody (U-CyTech Utrecht, The Netherlands), Streptavidin-Alkaline Phosphatase conjugated (BD Biosciences, NJ) and finally with BCIP/NBT 1-Step solution (Thermo Fisher Scientific, Rockford, IL). Plates were acquired and analyzed by an A.EL.VIS (Automated ELisa-spot assay Video System, Hannover, Germany) automated plate reader. The ELISpot response was considered positive when all of the following conditions were met: IFNy production present in Con-A stimulated wells; at least 50 specific spots/million splenocytes present; the number of spots seen in positive wells was three times higher than the number detected in the mock control wells (DMSO); and decreased responses with cell dilutions were seen. ELISpot data were expressed as IFNy spot forming cells (SFC) per million splenocytes.
Intracellular Cytokine Staining (ICS) and FACS analysis: ICS was performed by the following method , in Experiment 1 at 4 weeks post-vaccination, and in Experiment 2 at week 10 (2 weeks post 2nd boost).
Briefly, 2 x 106 mouse splenocytes were stimulated at 37°C in 5% C02 for 15-20 hours using a pool of overlapping 15-mer MAGEA3 peptides as antigen at a final concentration of 2μg/ml each peptide in the presence of Golgi-plug (according to the manufacturer's
instructions; Becton Dickinson Biosciences, NJ). DMSO (Sigma-Aldrich, Milan, Italy) was used as a negative control and PMA/Ionomycin (Sigma-Aldrich, Milan, Italy) were used as positive controls. After overnight stimulation, mouse splenocytes were incubated with purified anti-mouse CD16/CD32 clone 2.4G2 (Fc block: BD Biosciences, NJ) and then stained in FACS buffer (PBS, 1% FCS) with the following surface antibodies: APC anti-mouse CD3e, clone 145-2C11; PE anti-mouse CD4, clone L3T4; PerCP anti-mouse CD8a, clone 53-6.7 (all from BD Biosciences, NJ).
Intracellular staining was performed after treatment with Cytofix/Cytoperm and in the presence of PermWash (BD Biosciences, NJ) using FITC anti-mouse IFNy, clone XMG1.2 (BD
Biosciences, NJ). Stained cells were acquired on a FACS Canto flow cytometer and analyzed using DIVA software (BD Biosciences, NJ).
CD8-Antigen specific T cells by MAGEA3 pentamer staining: Analysis of CD 8 -antigen specific T cells was performed by the following pentamer staining method, in Experiment 1 using individual blood samples taken from mice at two weeks and four weeks post vaccination; and for Experiment 2 using blood samples taken at week two, week four (pre-boost), week 5, week 8, week 9, and week 10 (pooled blood samples from each group were tested, except for samples taken at week 5 and week 9 when individual samples were tested).
Blood (about 400 μΐ) was taken from each mouse via the retro-orbital sinus with a Pasteur pipette and collected in tubes containing Heparin as anticoagulant. Two steps of red blood cell lysis were performed incubating the samples with ACK lysing buffer (Gibco) for 15 minutes.
Cells were washed with PBS/0.1% BSA and stained with ^g H2Kd-MAGEA3 (SYVKVLHH;
SEQ ID NO: 3) pentamer PE-conjugated (Proimmune) for 20min at room temperature (RT), followed first by LIVE/DEAD® Fixable Violet Staining (Molecular Probes), then staining in FACS buffer (PBS, 1% FCS) with the following surface antibodies: APC anti-mouse CD3e, clone 145-2C11; PerCP anti-mouse CD8a, clone 53-6.7, anti-mouse CD19 (all from BD
Biosciences, NJ). Stained cells were acquired on a FACS Canto flow cytometer and analyzed using DIVA software (BD Biosciences, NJ).
RESULTS: Comparison of Co-administered schedules in Mice
As described in Example 1, The immunogenicity of co-administered PanAd3-MAGEA3 and recMAGEA3 AS15-adjuvanted protein (groups 3-5) was compared to the immunogenicity of a single injection of adenovirus vector alone (Group 1), contralateral coadministration of adenovirus vector (Group 2), and a single injection of recMAGEA3 AS15-adjuvanted protein alone (Group 6).
Results were obtained by pentamer staining on whole blood at week 2 and week 4 (individual mice, shown in Figure 1) and IFNy ELISpot-ICS on spleen at week 4 (individual mice, Figure 2).
Figure 1. Comparing Groups 3-5 (coadministered PanAd3-MAGEA3 and recMAGEA3
AS15-adjuvanted protein), the percentage of MAGEA3 -specific CD8 cells is much greater at both time points in Group 3 (contralateral co-administration of PanAd3-MAGEA3 and recMAGEA3 AS15-adjuvanted protein) compared to co-administration in the same limb of Pan Ad3 -MAGE A3 and recMAGEA3 AS15-adjuvanted protein (Group 4), and to co-formulated coadministration of Pan Ad3 -MAGE A3 and recMAGEA3 AS15-adjuvanted protein (Group 5). These results indicate there is an advantage in administering the two components (adenoviral vector and polypeptide immunogen) contralaterally.
Figure 2 shows the % of MAGEA3 specific CD4 cells as well as CD8 cells, measured at week 4 post injection. (This analysis was not done for Groups 5 and 6).
RESULTS: MAGEA3 prime/boost with viral vectors and ASCI
The immunogenicity of sequential administrations was evaluated, using different administration schedules. Administration was as shown in TABLE 2. Results of Example 2 are as follows.
MAGEA3 specific CD8 responses (measured by pentamer staining using blood samples) are shown in Figure 3, where Groups 1-7 are shown from left to right across the graph. Data was generated with pooled blood samples from each group, except at week 5 and week 9 where individual samples were tested (Mean + SEM shown).
MAGEA3 specific CD8 responses (measured by ICS on samples of splenocytes at week ten (two weeks after the second boost)) are shown in Figure 4. Circles represent CD4 cells and squares represent CD8 cells; a horizontal line represents the geometric mean for each cell type per group. The data indicate that regimens based on contralateral administration of viral vector and polypeptide immunogen induced potent CD4 and CD8 T cell responses.
Sequences
SEQ ID NO: l (human MAGE- A3 protein)
MPLEQRSQHC KPEEGLEARG EALGLVGAQA PATEEQEAAS SSSTLVEVTL GEVPAAESPD PPQSPQGASS LPTTMNYPLW SQSYEDSSNQ EEEGPSTFPD LESEFQAALS RKVAELVHFL
LLKYRAREPV TKAEMLGSVV GNWQYFFPVI FSKASSSLQL VFGIELMEVD PIGHLYI FAT
CLGLSYDGLL GDNQIMPKAG LLIIVLAIIA REGDCAPEEK IWEELSVLEV FEGREDSILG
DPKKLLTQHF VQENYLEYRQ VPGSDPACYE FLWGPRALVE TSYVKVLHHM VKISGGPHIS
YPPLHEWVLR EGEE -314 (SEQ ID NO : 1 )
SEQ ID NO:2 (recMAGEA3; fusion protein of Protein D fragment + MAGEA3 amino acids 3- 314)
MDPKTLALSL LAAGVLAGCS SHSSNMANTQ MKSDKI I IAH RGASGYLPEH - 50
TLESKALAFA QQADYLEQDL AMTKDGRLW IHDHFLDGLT DVAKKFPHRH -100
RKDGRYYVID FTLKEIQSLE MTENFETMDL EQRSQHCKPE EGLEARGEAL -150
GLVGAQAPAT EEQEAASSSS TLVEVTLGEV PAAESPDPPQ SPQGASSLPT -200
TMNYPLWSQS YEDSSNQEEE GPSTFPDLES EFQAALSRKV AELVHFLLLK -250 YRAREPVTKA EMLGSWGNW QYFFPVIFSK ASSSLQLVFG IELMEVDPIG -300
HLYIFATCLG LSYDGLLGDN QIMPKAGLLI IVLAI IAREG DCAPEEKIWE -350
ELSVLEVFEG REDSILGDPK KLLTQHFVQE NYLEYRQVPG SDPACYEFLW -400
GPRALVETSY VKVLHHMVKI SGGPHISYPP LHEWVLREGE EGGHHHHHHH*-450 SEQ ID NO: 3 (MAGEA3 peptide): SYVKVLHH
SEQ ID NO:4 (CD 8 MAGEA3 immunodominant peptide) : SYVKVLHHM
Claims
1. A method of raising an immune response in a subject, comprising administering (a) a polypeptide immunogen and (b) a polynucleotide vector encoding said polypeptide immunogen, where said polypeptide immunogen and said polynucleotide-based vector are administered at about the same time, in different limbs of said subject.
2. A method of treating cancer in a subject in need thereof, comprising administering (a) a polypeptide immunogen selected from a tumor-associated antigen, an immunogenic fragment of a tumor-associated antigen, and a fusion protein comprising a tumor-associated antigen or an immunogenic fragment of a tumor-associated antigen, and (b) a polynucleotide vector encoding a polypeptide consisting of or comprising the same polypeptide immunogen, where said polypeptide immunogen and said polynucleotide-based vector are administered at about the same time, in different limbs of said subject, and where said subject's cancer expresses said tumor-associated antigen.
3. A method of increasing an immune response in a subject against a polypeptide immunogen, comprising administering (a) a polypeptide immunogen and (b) a polynucleotide vector encoding a polypeptide consisting of or comprising the same polypeptide immunogen, where said polypeptide immunogen and said polynucleotide-based vector are administered at about the same time, in different limbs of said subject, and where said immune response is increased compared to that which would be achieved by administration of said polypeptide immunogen and said polynucleotide vector at about the same time to the same limb of the subject.
4. A composition comprising a polynucleotide vector encoding a polypeptide
immunogen, for use in raising an immune response in a subject by coadministration with a polypeptide immunogen, where said polynucleotide vector and said polypeptide immunogen are administered in different limbs of said subject at about the same time.
5. A composition comprising a polypeptide immunogen, for use in raising an immune response in a subject by coadministration with a composition comprising a polynucleotide vector encoding a polypeptide consisting of or comprising the same polypeptide immunogen,
where said polynucleotide vector and said polypeptide immunogen are administered in different limbs of said subject at about the same time.
6. Use of a composition comprising a polynucleotide vector encoding a polypeptide immunogen, in the manufacture of a medicament for use in raising an immune response in a subject by coadministration with a composition comprising a polypeptide immunogen, where said polynucleotide vector and said polypeptide immunogen are administered in different limbs of said subject at about the same time.
7. Use of a composition comprising a polypeptide immunogen, in the manufacture of a medicament for use in raising an immune response in a subject by coadministration with a composition comprising a polynucleotide vector encoding a polypeptide consisting of or comprising the same polypeptide immunogen, where said polynucleotide vector and said polypeptide immunogen are administered in different limbs of said subject at about the same time.
8. The method, composition or use of any one of claims 1-7 where said polypeptide immunogen is formulated as a pharmaceutical composition.
9. The method, composition or use of claim 8 where said pharmaceutical composition further comprises an adjuvant.
10. The method, composition or use of claim 9 where said adjuvant comprises MPL, QS21, or an immunostimulatory oligonucleotide.
11. The method, composition or use of claim 9 where said adjuvant is a liposomal formulation comprising QS21, monophosphoryl lipid A, and CpG7909.
12. The method, composition or use of claim 9 where said adjuvant is AS15.
13. The method, composition or use of any one of the preceding claims where said polypeptide immunogen is a tumor-associated antigen.
14. The method, composition or use of any one of the preceding claims where said polypeptide immunogen is MAGE-A3, an immunogenic fragment or fusion protein thereof.
15. The method, composition or use of any one of the preceding claims where said polypeptide immunogen is selected from: MAGEA3 protein having SEQ ID NO: l; a polypeptide having at least 95% identity to SEQ ID NO: 1; an immunogenic fragment of SEQ ID NO: 1; a polypeptide having SEQ ID NO:2; and a polypeptide having at least 95% identity to SEQ ID NO:2.
16 . The method, composition or use of any one of the preceding claims where said polynucleotide vector is selected from plasmid DNA, a viral vector, and a bacterial vector.
17. The method, composition or use of any one of the preceding claims where said polynucleotide vector is formulated as a pharmaceutical composition.
18. The method, composition or use of any one of the preceding claims where said polynucleotide vector is an adenoviral vector.
19. The method, composition or use of any one of the preceding claims where said polynucleotide vector encodes a tumor-associated antigen.
20. The method, composition or use of any one of the preceding claims where said polynucleotide vector encodes MAGE- A3, an immunogenic fragment or fusion protein thereof.
21. The method, composition or use of any one of the preceding claims where said subject is a mammal selected from humans, non-human primates, and rodents.
22. The method, composition or use of any one of the preceding claims where said subject is a human who has been treated for or diagnosed with a MAGE- A3 expressing tumor, and said polypeptide immunogen is a MAGE-A3 polypeptide, an immunogenic fragment of a MAGE- A3 polypeptide, or a fusion protein comprising a MAGE- A3 polypeptide or immunogenic fragment of a MAGE- A3 polypeptide.
23. The method, composition or use of claim 22 where said subject has, or has been treated for, a MAGE- A3 expressing tumor , and said polypeptide immunogen is MAGEA3;
24. The method, composition or use of any one of claims 22-23 where said MAGEA3 expressing tumor is melanoma, non-small cell lung cancer (NSCLC), bladder cancer, or hepatic cancer.
25. The method, composition or use of any one of the preceding claims further comprising a subsequent administration of (a) a polypeptide immunogen and (b) a polynucleotide vector encoding a polypeptide consisting of or comprising the same polypeptide immunogen, where said polypeptide immunogen and said polynucleotide-based vector are administered at about the same time, in different limbs of said subject, and where said subsequent administration occurs within six months of the initial administration.
26. A kit comprising a polynucleotide vector encoding an immunogenic polypeptide, for use in raising an immune response in a subject when coadministered with a pharmaceutical composition comprising an immunogenic polypeptide, where said coadministration occurs in different limbs of said subject.
27. A kit comprising (a) a polynucleotide vector encoding an immunogenic polypeptide and (b) a composition comprising an immunogenic polypeptide, for use in raising an immune response in a subject when said polynucleotide vector and said immunogenic polypeptide are coadministered to different limbs of said subject.
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