Her2/neu immunogenic composition
11154599 · 2021-10-26
Assignee
Inventors
- Geert Mudde (Breitenfurt, AT)
- Jorge Sepulveda (Vienna, AT)
- Christopher Taus (Vienna, AT)
- Liesbeth Mudde-Boer (Breitenfurt, AT)
Cpc classification
A61K39/39
HUMAN NECESSITIES
A61K2039/55561
HUMAN NECESSITIES
International classification
A61K39/00
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
Abstract
The invention provides for an immunogenic composition comprising a. a directed adjuvant comprising at least an anti-CD32 moiety linked to a TLR9 ligand and a first peptidic alpha-helix; and b. an immunogen comprising an extracellular Her2/neu domain that is linked to a second peptidic alpha-helix coiled to the first alpha-helix; The invention further provides a kit for producing such immunogenic composition, a vaccine comprising such immunogenic composition and its medical use, such as for treating Her2/neu positive tumor diseases.
Claims
1. An immunogenic composition comprising: (a) a directed adjuvant comprising an anti-CD32 binding moiety linked to a TLR9 ligand and a first peptidic alpha-helix, wherein the anti-CD32 binding moiety is an anti-CD32 antibody or an antigen-binding fragment thereof, and wherein the TLR9 ligand is a TLR9 agonist selected from the group consisting of a CpG oligodeoxynucleotide class A molecule, a CpG oligodeoxynucleotide class B molecule, and a CpG oligodeoxynucleotide class C molecule; and (b) at least one immunogen comprising an extracellular Her2/neu domain monomer that is linked to a second peptidic alpha-helix that is bound to the first alpha-helix to form a coiled coil, wherein the extracellular Her2/neu domain monomer comprises: (i) a polypeptide identified by the amino acid sequence SEQ ID NO:1 or SEQ ID NO:2; or (ii) a polypeptide with at least 90% sequence identity to the polypeptide of subparagraph (i); wherein the first and second peptidic alpha-helices each comprise 2-9 repeats of a matching amino acid sequence selected from the group consisting of SEQ ID NOs:11, 12, and 15-20.
2. The immunogenic composition of claim 1, comprising at least two immunogens linked to the second peptidic alpha-helix.
3. The immunogenic composition of claim 1, wherein each of said first and second alpha-helices specifically bind to each other with a Kd of less than 10.sup.−6 M.
4. The immunogenic composition of claim 1, comprising one or more linker sequences composed of glycine, serine, and/or lysine residues.
5. The immunogenic composition of claim 1, wherein the immunogen comprises the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.
6. A vaccine comprising the immunogenic composition of claim 1 and a pharmaceutically acceptable carrier.
7. A kit for preparing the immunogenic composition of claim 1, comprising the following components: (a) a directed adjuvant comprising an anti-CD32 binding moiety linked to a TLR9 ligand and a first peptidic alpha-helix, wherein the anti-CD32 binding moiety is an anti-CD32 antibody or an antigen-binding fragment thereof, and wherein said TLR9 ligand is a TLR9 agonist selected from the group consisting of a CpG oligodeoxynucleotide class A molecule, a CpG oligodeoxynucleotide class B molecule, and a CpG oligodeoxynucleotide class C molecule; and (b) an immunogen comprising an extracellular Her2/neu domain monomer that is linked to a second peptidic alpha-helix coiled to the first alpha-helix, wherein the extracellular Her2/neu domain comprises or consists of: (i) a polypeptide identified by the amino acid sequence SEQ ID NO:1 or SEQ ID NO:2; or (ii) a polypeptide with at least 90% sequence identity to the polypeptide of subparagraph (i); wherein the first and second peptidic alpha-helices each comprise 2-9 coil repeats of a matching amino acid sequence selected from the group consisting of SEQ ID NOs:11, 12, and 15-20, and wherein the first peptidic alpha-helix is capable of binding to the second peptidic alpha-helix to form a coiled coil.
8. A method of treating a subject suffering from Her2/neu positive tumor diseases, comprising administering the immunogenic composition of claim 1 to the subject.
9. The method of claim 8, wherein the composition is administered to the subject in an effective amount employing a prime-boost strategy.
10. The method of claim 8, wherein the composition is administered to the subject in an effective amount ranging between 0.0001 and 2 mg per administration.
11. The method of claim 8, wherein the subject is further treated by chemotherapy, immunotherapy, inhibitor therapy and/or radiotherapy.
12. The method of claim 8, wherein a protective immune response is triggered in the subject with a serum IgG titer against Her2/neu of at least 1/100.
13. The immunogenic composition of claim 1, wherein said Her2/neu domain consists of a polypeptide identified by the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
14. The immunogenic composition of claim 2, comprising 3 or 4 immunogens linked to the second peptidic alpha-helix.
15. The immunogenic composition of claim 1, wherein said anti-CD32 binding moiety binds to CD32a.
16. The immunogenic composition of claim 4, wherein the linker sequences comprise the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5.
17. The method of claim 8, wherein the subject is suffering from a disease selected from the group consisting of breast cancer, ovarian cancer, stomach cancer, uterine cancer, colorectal cancer and non-small cell lung cancer.
Description
FIGURES
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DETAILED DESCRIPTION OF THE INVENTION
(6) Specific terms as used throughout the specification have the following meaning.
(7) The term “adjuvant” as used herein shall mean an integrated or co-administered component of a vaccine, which: enhances the immune response to a specific immunogen, e.g. an antigen or a hapten. The immune response is typically greater than the immune response elicited by an equivalent amount of the immunogenic composition administered without the adjuvant,
(8) and/or the adjuvant is used to direct a particular type or class of immune response against the immunogen, e.g. a Th1 or Treg type of immune response, herein understood as “directed adjuvant”.
(9) An “effective amount” of an adjuvant of the present invention specifically is an amount which enhances an immunological response to the immunogen such that, for example, lower or fewer doses of the immunogenic composition are required to generate an efficient immune response of the intended class.
(10) The directed adjuvant according to the invention not only mediates the efficient immune response, but also the regulation of the immune response in the desired way. By directing the immunogen to the appropriate immune cells for its internalization and further processing, the Th1 immune response is induced rather than the Th2 immune response, in particular when employing a TLR9 ligand that is a TLR9 agonist of group 3. If a TLR9 agonist of group 1 is combined with an anti-CD32 moiety that binds CD32b, the induction of Treg cells is usually anticipated.
(11) An “effective amount” of an immunogenic composition, e.g. as used in a vaccine of the invention refers to an amount sufficient to show a meaningful benefit in a subject being treated, when administered as part of a vaccination dosing regimen. Those of ordinary skill in the art will appreciate that, in some embodiments, a particular composition may be considered to contain a prophylactically or therapeutically effective amount if it contains an amount appropriate for a unit dosage form administered in a specific dosing regimen, even though such amount may be insufficient to achieve the meaningful benefit if administered as a single unit dose. Those of ordinary skill will further appreciate that an effective amount of an immunogenic composition may differ for different subjects receiving the composition, for example depending on such factors as the desired biological endpoint, the nature of the composition, the route of administration, the health, size and/or age of the subject being treated, etc. In some embodiments, an effective amount is one that has been correlated with beneficial effect when administered as part of a particular dosing regimen, e.g. a single administration or a series of administrations such as in a “boosting” regimen.
(12) The term “peptidic alpha-helix” as used herein shall mean a coiled structural motif based on a peptide sequence comprising a number of repeats, also called coil repeats. Such alpha-helix is capable of binding to another counterpart, also called matching alpha-helix of the same type to form a dimer, trimer or further oligomer, also called coiled coil.
(13) A coiled coil is a structural motif in polypeptides or peptides, in which two to seven alpha-helices are coiled together like the strands of a rope. In some embodiments, the coiled coil of the vaccine is one with two alpha-helices coiled together. Such alpha helical regions are likely to form coiled-coil structures and may be involved in oligomerization of the coil repeats as measured in a suitable coiled coil interaction binding assay.
(14) Specifically a dimer of alpha-helices can be formed by contacting the two monomers, such that the dimer is formed through an interaction with the two alpha helix coiled coil domains. In some embodiments the coils comprise a peptide with the amino acid sequence as set forth in SEQ ID NO: 11 or 12 (coil and anti-coil), which include x repeats.
(15) TABLE-US-00004 (SEQ ID 11) EVSAL E5: (SEQ ID 13) EVSALEKEVSALEKEVSALEKEVSALEKEVSALEK-NH2 (SEQ ID 12) KVSAL K5: (SEQ ID 14) KVSALKEKVSALKEKVSALKEKVSALKEKVSALKE-NH2
(16) Alternatively, any of the sequences described by Chao et al..sup.16 or Litowsky et al..sup.17;18 or functional equivalents, which generate the specific coiled-coil type linkage, may be used, such as indicated in the following table, including variants thereof, e.g. with a different number of repeats, or one, two or three point mutations in the coil type:
(17) TABLE-US-00005 Coil Type Type/number of repeats: Exemplary sequence EIAAL E3: EIAALEKEIAALEKEIAALEK-NH2 (SEQ ID 21) (SEQ ID 15) EIAAL E4: EIAALEKEIAALEKEIAALEKEIAALEK-NH2 (SEQ ID 22) (SEQ ID 15) KIAAL K3: KIAALKEKIAALKEKIAALKE-NH2 (SEQ ID 23) (SEQ ID 16) KIAAL K4: KIAALKEKIAALKEKIAALKEKIAALKE-NH2 (SEQ ID 24) (SEQ ID 16) EISAL E3: EISALEKEISALEKEISALEK-NH2 (SEQ ID 25) (SEQ ID 17) EISAL E4: EISALEKEISALEKEISALEKEISALEK-NH2 (SEQ ID 26) (SEQ ID 17) KISAL K3: KISALKEKISALKEKISALKE-NH2 (SEQ ID 27) (SEQ ID 18) KISAL K4: KISALKEKISALKEKISALKEKISALKE-NH2 (SEQ ID 28) (SEQ ID 18) EVAAL E3: EVAALEKEVAALEKEVAALEK-NH2 (SEQ ID 29) (SEQ ID 19) EVAAL E4: EVAALEKEVAALEKEVAALEKEVAALEK-NH2 (SEQ ID 30) (SEQ ID 19) KVAAL K3: KVAALKEKVAALKEKVAALKE-NH2 (SEQ ID 31) (SEQ ID 20) KVAAL K4: KVAALKEKVAALKEKVAALKEKVAALKE-NH2 (SEQ ID 32) (SEQ ID 20) EVSAL E3: EVSALEKEVSALEKEVSALEK-NH2 (SEQ ID 33) (SEQ ID 11) EVSAL E4: EVSALEKEVSALEKEVSALEKEVSALEK-NH2 (SEQ ID 34) (SEQ ID 11) KVSAL K3: KVSALKEKVSALKEKVSALKE-NH2 (SEQ ID 35) (SEQ ID 12) KVSAL K4: KVSALKEKVSALKEKVSALKEKVSALKE-NH2 (SEQ ID 36) (SEQ ID 12)
(18) For the purpose of the invention, the preferred type of a coiled coil is a dimer, either a heterodimer (heterocoil) of two different, but matching helices, which differ in at least one amino acid in the coil repeat sequence, or else a homodimer of two identical matching helices, i.e. those comprising the matching coil repeat sequences (the “coils”).
(19) Specifically, the number of coil repeats is 2-9, specifically 3-5, preferably any of the combinations 3+3, 3+4, 3+5, 4+4, 4+5, 5+5, 4+3, 5+3 or 5+4.
(20) As an alternative to heptad repeats (repeats of an amino acid sequence consisting of 7 amino acids, 7-mers), 3-mers, 4-mers, 5-mers, 6-mers, 8-mers, 9-mers, or 10-mers may be used.
(21) In case of a homodimeric coiled coil, the typical number of coil repeats is specifically not more than 5, so to avoid undesired mismatches of the structure. In case of a heterodimeric coiled coil, it is typically desirable to employ a length of the peptide sequence with at least 3 coils. Thereby the binding of the components of the vaccine, i.e. the directed adjuvant and the immunogen components, to each other is typically achieved with preferred high affinity of a Kd of less than 10.sup.−7 M, more preferred less than 10.sup.−8 M or 10.sup.−9 M. However, although more repeats increase the affinity, this may be at the cost of increased homodimerisation.
(22) The components of the immunogenic composition of the invention may also comprise a peptide spacer so to link the anti-CD32 moiety and/or the TLR9 ligand, and optionally also the epitopes (of the Her2/neu domain) with the coil repeats, respectively. For example, the peptide spacer can be on either or both ends of a coiled coil. Each of the peptide spacers can be attached to a single alpha helix coiled coil domain of the coiled coil.
(23) The peptide spacer can be, for example, a peptide of 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids or more, either linear or branched, e.g. to provide for two, three, four, or more branches. The number of amino acids in the peptide spacer may be, in some embodiments, 20 amino acids or up to 10 amino acids greater or fewer, depending on the particular sequences and length of the coil.
(24) The term “anti-CD32 moiety” as used herein shall mean a ligand specifically binding to the cellular target CD32, either CD32a, CD32b or both, CD32a and CD32b. The moiety can be any binding structure, such as derived from proteins, polypeptides or peptides, including antibodies and antibody fragments or composite molecules with a binding part. The binding part of the molecules or molecule complex of the invention can be comprised of proteins such as antibodies or antibody fragments, such as Fab, Fv, scFv, dAb, F(ab)2, minibody, small mutated immunoglobulin domains, or other biological binders, such as soluble T-cell receptor, Darpins, etc. Antibodies and antibody fragments and derivatives may be generated and selected for binding to CD32 according to known methods such as hybridoma technology, B-cell cloning, phage display, ribosome display or cell surface display of antibody libraries, array screening of variant antibodies. Exemplary anti-CD32 moieties are scFv derived from the anti CD32 monoclonal antibody AT-10.sup.19, IV.3.sup.20, 2E1.sup.21 or any other aCD32 monoclonal antibody.
(25) The specific binding may be determined in a suitable binding assay, such as conventional immunoassays. There are numerous methods known in the art for detecting binding in an immunoassay. Various immunoassays known in the art can be used including competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, western blot, BIAcore etc.
(26) The term “cross-reactive” with respect to antigens or antibodies as used herein shall refer to epitopes shared between antigens of different origin, e.g. from human, rhesus monkey or mouse origin. The N-terminal epitope consisting or comprising the first 4 AA of G17 was found to be cross-reactive in peptides of various origin, which epitope triggers an immune response and IgG antibodies that are cross-reactive with the epitopes.
(27) The immunogenic composition of the invention is specifically useful to treat Her2/neu positive tumor diseases or disease conditions in which Her2/neu overexpression is playing a role.
(28) The term “Her2/neu positive tumor” as used herein shall refer to tumors or disease or disease conditions associated therewith, of e.g. breast, ovarian, stomach, uterine, colorectal and non-small cell lung cancer. The term is specifically used herein with regard to treating the tumor for preventing tumor disease progression, for a positive tumor response or for tumor shrinkage. The term is also applied to minimal residual disease, which would be successfully treated, e.g. targeting circulating tumor cells to reduce their number below a certain threshold, e.g. below the detection limit.
(29) The term “Her2/neu domain” or “Extracellular Her2/neu domain” as used herein shall refer to the Her2/neu protein including the extracellular domain of Her2/neu, or at least part of it. Specifically, the Her2/neu domain may or may not comprise the intracellular domain, and optionally comprises at least the T-cell epitopes of the intracellular domain. Preferably, the Her2/neu domain does not comprise the transmembrane domain.
(30) The Her2/neu domain may be of human origin, or other mammalian origin, including rhesus monkey, or mouse, thus, has a human or other mammalian sequence, or may be an artificial construct, such as to incorporate artificial sequences, e.g. obtained by changing the type and/or sequence of amino acid residues in the native (naturally occurring) sequence. The term shall specifically include variants of human Her2/neu domain, with an amino acid sequence SEQ ID 1 or SEQ ID 2, or sequences with a certain sequence identity thereto, or fragments thereof, e.g. a fragment or hybrid polypeptide combining or fusing at least two fragments of the Her2/neu domain (to obtain a hybrid), but differs from its amino acid sequence, in that it is derived from a homologous sequence of a different species. These are referred to as naturally occurring variants or analogs. The term “analogs” shall also refer to chimeric constructs originating from two or more origins, wherein at least one part is naturally-occurring, e.g. which constitutes the major part (at least 50%) of the Her2/neu domain, and another part is different thereto, either naturally occurring or non-naturally occurring (synthetic or artificial).
(31) The term shall specifically include fragments or functionally active variants of the Her2/neu domain, e.g. those comprising one or more point mutations, or else peptides or polypeptides comprising further amino acid sequences besides the Her2/neu domain, e.g. by extending the N-terminus and/or the C-terminus by additional one or more amino acid residues or sequences. An extension of the C-terminus is e.g. preferred with repeats of Her2/neu domain sequences, either identical or not, or with further amino acid sequences of the Her2/neu protein.
(32) The term “functionally active variants” as used herein with respect to the Her2/neu domain as described herein, shall mean a sequence resulting from modification of this sequence (a parent sequence), e.g. by insertion, deletion or substitution of one or more amino acids, such as by recombination techniques or chemical derivatization of one or more amino acid residues in the amino acid sequence, or nucleotides within the coding nucleotide sequence, or at either or both of the distal ends of the sequence, and which modification does not affect (in particular impair) the activity of this sequence. In the case of a Her2/neu domain eliciting a certain immune response to target Her2/neu, the functionally active variant of the Her2/neu domain would still incorporate the antigenic determinant or epitope, though this could be changed, e.g. to increase the immunogenicity. Specifically, the functionally active variants of the Her2/neu domain have the potency to elicit IgG anti-gastrin antibodies in a treated subject, which antibodies cross-react with the endogenous Her2/neu receptor on the surface of tumor cells. Functionally active variants can further be characterized by eliciting the desired T-cell immune response. Including CD4+ Th cells and/or CD8+ Tc cell which may be directed against any part of the Her2/neu protein either the intracellular or the extra cellular domain thus providing the necessary T cell help for antibody production of activation of the CD8 cells which may directly kill the Her2/neu expressing tumor cell
(33) Functionally active variants may be obtained, e.g. by changing the sequence of a parent Her2/neu domain, e.g. the human, rhesus monkey or murine Her2/neu domain, or a fragment thereof, by introducing one or more modifications that do not substantially impair the epitopes, to obtain a molecule with substantially the same immunogenicity. The term “substantially the same immunogenicity” as used herein refers to the amount of an immune response or anti-Her2/neu IgG antibodies induced in a subject treated with the immunogenic composition, which amount is preferably at least 20% at least 30% at least 40%, at least 50% at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or even at least 100% or at least 110%, or at least 120%, or at least 130%, or at least 140%, or at least 150%, or at least 160%, or at least 170%, or at least 180%, or at least 190%, e.g. up to 200% of the amount as determined for the parent polypeptide.
(34) In a preferred embodiment the functionally active variant of a parent polypeptide
(35) a) is derived from the polypeptide by at least one amino acid substitution, insertion (addition) and/or deletion, e.g. comprising one or more point mutations wherein the functionally active variant has a specific sequence identity to the parent molecule, such as at least 50% sequence identity, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, still more preferably at least 90%; and/or
(36) b) consists of the polypeptide and additionally at least one amino acid heterologous to the polypeptide.
(37) Functionally active variants may be obtained by sequence alterations in the polypeptide sequence, e.g. by one or more point mutations, wherein the sequence alterations substantially retains a function of the unaltered polypeptide sequence, when used in according to the invention. Such sequence alterations or point mutations can include, but are not limited to, (conservative) substitutions, additions, deletions, mutations and insertions, e.g. the alteration of 1, 2, 3, or 4 amino acids, or by addition or insertion of one to several amino acids, e.g. 1, 2, 3, or 4 amino acids, or by a chemical derivatization of one to several amino acids, e.g. 1, 2, 3, or 4, or combination thereof, preferably by point mutations that are not contiguous. The substitutions in amino acid residues may be conservative substitutions, for example, substituting one hydrophobic amino acid for an alternative hydrophobic amino acid.
(38) Conservative substitutions are those that take place within a family of amino acids that are related in their side chains and chemical properties. Examples of such families are amino acids with basic side chains, with acidic side chains, with non-polar aliphatic side chains, with non-polar aromatic side chains, with uncharged polar side chains, with small side chains, with large side chains etc.
(39) Preferred point mutations refer to the exchange of amino acids of the same polarity and/or charge. In this regard, amino acids refer to twenty naturally-occurring amino acids encoded by sixty-four triplet codons. These 20 amino acids can be split into those that have neutral charges, positive charges, and negative charges:
(40) The “neutral” amino acids are shown below along with their respective three-letter and single-letter code and polarity:
(41) Alanine: (Ala, A) nonpolar, neutral;
(42) Asparagine: (Asn, N) polar, neutral;
(43) Cysteine: (Cys, C) nonpolar, neutral;
(44) Glutamine: (Gln, Q) polar, neutral;
(45) Glycine: (Gly, G) nonpolar, neutral;
(46) Isoleucine: (Ile, I) nonpolar, neutral;
(47) Leucine: (Leu, L) nonpolar, neutral;
(48) Methionine: (Met, M) nonpolar, neutral;
(49) Phenylalanine: (Phe, F) nonpolar, neutral;
(50) Proline: (Pro, P) nonpolar, neutral;
(51) Serine: (Ser, S) polar, neutral;
(52) Threonine: (Thr, T) polar, neutral;
(53) Tryptophan: (Trp, W) nonpolar, neutral;
(54) Tyrosine: (Tyr, Y) polar, neutral;
(55) Valine: (Val, V) nonpolar, neutral; and
(56) Histidine: (His, H) polar, positive (10%) neutral (90%).
(57) The “positively” charged amino acids are:
(58) Arginine: (Arg, R) polar, positive; and
(59) Lysine: (Lys, K) polar, positive.
(60) The “negatively” charged amino acids are:
(61) Aspartic acid: (Asp, D) polar, negative; and
(62) Glutamic acid: (Glu, E) polar, negative.
(63) “Percent (%) amino acid sequence identity” with respect to the polypeptide sequences described herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific polypeptide sequence, after aligning the sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
(64) Functionally active variants may be obtained by any of the known mutagenesis methods, including point mutations at desired positions, e.g. obtained by randomisation techniques. In some cases positions are chosen randomly, e.g. with either any of the possible amino acids or a selection of preferred amino acids to randomise the polypeptide sequences. In this regard, the term “mutagenesis” refers to any art recognized technique for altering a polynucleotide or polypeptide sequence.
(65) The term “immunogen” as used herein shall mean an antigen or immunogen of polypeptide or peptidic structure, in particular an immunogen that comprises or consists of a polypeptide or peptide of a specific amino acid sequence, which is either provided as a linear polypeptide (peptide) or branched polypeptide (e.g. a synthetic polypeptide or peptide), comprising naturally occurring amino acid residues or modified ones, e.g. a derivative obtained by modification or chemical derivatization, such as by phosphorylation, methylation, acetylation, amidation, formation of pyrrolidone carboxylic acid, isomerization, hydroxylation, sulfation, flavin-binding, cysteine oxidation and nitrosylation.
(66) The immunogen or Her2/neu domain as used herein is specifically designed to trigger an immune response in a subject, and particularly includes one or more antigenic determinants, which can be possibly recognized by a binding site of an antibody or is able to bind to the peptide groove of HLA class I or class II molecules or other antigen presenting molecules such as CD1 and as such may serve as stimulant for specific T cells. The target antigen is either recognized as a whole target molecule or as a fragment of such molecule, especially substructures, e.g. a polypeptide or carbohydrate structure of targets, generally referred to as “epitopes”, e.g. B-cell epitopes, T-cell epitope, which are immunologically relevant, i.e. are also recognizable by natural or monoclonal antibodies. Herein the use of B cell epitopes and T cell epitopes is specifically preferred.
(67) The term “epitope” as used herein according to the present invention shall in particular refer to a molecular structure which may completely make up a specific binding partner or be part of a specific binding partner to a binding site of an antibody. Chemically, an epitope of a Her2/neu domain of the present invention may be a peptide epitope that usually includes at least 3 amino acid residues, preferably 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 amino acids. There is no critical upper limit to the length of the peptide, which could comprise even the full length of an amino acid sequence of a protein.
(68) The Her2/neu domain of the invention is specifically understood as a self-antigen. The term “self-antigen” as used herein means any antigen, specifically polypeptide or peptide produced by a normal, healthy subject that does not elicit an immune response as such. These self-antigens may be produced at aberrant or high levels in certain disease states, including cancer disease, so called tumour associated antigens (TAAs). Herein, the Her2/neu is understood as a self-antigen in human subjects, and specifically as a TAA in subjects suffering from a Her2/neu positive (or overexpressing) tumor.
(69) It is understood that the self-antigens such as used according to the invention, can be naturally-occurring, recombinantly or synthetically produced. It is also understood that the self-antigens need not be identical to the naturally produced antigen, but rather can include variations thereto having certain sequence identities, similarities or homology.
(70) The Her2/neu domain or the immunogenic composition used in the vaccine as described herein, is usually contained in a vaccine in an effective amount, which is herein specifically understood as “immunologically effective amount”. By “immunologically effective amount”, it is meant that the administration of that amount to a subject, either in a single dose or as part of a series of doses, is effective on the basis of the therapeutic or prophylactic objectives. This amount will vary depending upon the health and physical condition of the subject to be treated, age, the capacity of the subject's immune system to synthesize antibodies, the degree of immune response desired, the formulation of the vaccine, and other conditions.
(71) The invention also provides a method for treating a subject or raising an immune response in a subject, comprising the step of administering an immunologically effective amount of the peptide immunogen, the immunogenic composition or the vaccine of the invention.
(72) An effective amount or dosage may range from 0.0001 to 2 mg, e.g. between 0.001 and 2 mg, of the immunogenic composition administered to the subject in need thereof, e.g. an adult human subject. The effective dosage of the immunogenic composition is capable of eliciting an immune response in a patient of effective levels of antibody titer to bind to endogenous Her2/neu, e.g. 1-3 months after immunization. The effectiveness of the therapy may be assayed by the Her2/neu antibody titers in samples of blood taken from the subject, or by measuring tumor load using classical clinical methods to monitor the presence of absence of individual tumors.
(73) The term “TLR9 ligand” as used herein is understood in the following way.
(74) Toll-like receptor 9 (TLR9) recognizes unmethylated bacterial CpG DNA and initiates a signalling cascade leading to the production of proinflammatory cytokines. There are numerous structures or sequences that have been shown to act as a ligand of TLR9, i.e. bind to this receptor and thereby either activate (stimulate, upregulate, TLR9 agonist) or de-activate (downregulate, TLR) antagonist) TLR9. For instance, microbial DNA or synthetic DNA, e.g. synthetic CpG ODN may stimulate TLR9 with variations in the number and location of CpG dimers, as well as the precise base sequences flanking the CpG dimers. Synthetic CpG ODN differ from microbial DNA in that they have a partially or completely phosphorothioated backbone instead of the typical phosphodiester backbone and may or may not have a poly G tail at the 3′ end, 5′ end, or both.
(75) The term “agonist” in conjunction with the TLR9 ligand as used herein shall specifically refer to the binding and activation of TLR9 in a cell-based assay.
(76) The TLR9 ligand which is composed of a nucleotide sequence is typically coupled to the directed adjuvant component of the present immunogenic composition by chemical coupling e.g. using the commercially available Kit from Solulink. A peptidic TLR9 ligand may be coupled using standard peptide chemistry or may be integrated using recombinant DNA technology.
(77) Exemplary TLR9 ligands are ODN 2216.sup.22 (group 1), ODN 2006/ODN 2007.sup.15 (group2) and CpG-M362.sup.13 (group 3).
(78) Further exemplary TLR9 ligands may be peptides that mimic the action of a CpG TLR9 agonist, e.g. identified by or obtained from a peptide library, which are selected for the affinity to bind the TLR9 and proven agonistic activity, or protein ligands, including specific antibodies.
(79) The function of a TLR9 ligand or agonist may be determined in a suitable assay, e.g. in the following way: pDCs are purified from blood of a healthy donor as described by Tel et al..sup.23 and subsequently incubated with the appropriate concentration of the TLR9 ligand. After 24 h IFNa is measured in the supernatant using standard ELISA protocols. For determination of the maturation state of the cells, pDCs are stained for expression of CD80 CD83 or CD86 using standard FACS procedures with commercially available specific antibodies before and after the incubation with the TLR9 ligand.
(80) The number of reactive T cells that are activated upon exposure to the vaccine according to the invention may be determined by a number of methods including ELISPOT, FACS analysis, cytokine release, or T cell proliferation assays.
(81) As used herein, the term “specificity” or “specific binding” refers to a binding reaction which is determinative of the cognate ligand of interest in a heterogeneous population of molecules. Thus, under designated conditions (e.g. immunoassay conditions), one or more antigens are specifically bound by the respective binding site(s) of a binder, which does not bind in a significant amount to other molecules present in a sample. The specific binding means that binding is selective in terms of target identity, high, medium or low binding affinity or avidity, as selected. Selective binding is usually achieved if the binding constant or binding dynamics is at least 10 fold different, preferably the difference is at least 100 fold, and more preferred a least 1000 fold.
(82) The term “treatment” as used herein shall always refer to treating subjects for prophylactic (i.e. to prevent infection and/or disease status) or therapeutic (i.e. to treat diseases regardless of their pathogenesis) purposes. Treatment of a subject will typically be therapeutic in cases of cancer disease conditions, including Her2/neu positive tumors or Her2/neu overexpressing cancer. However, in case of patients suffering from a primary disease, which are at risk of disease progression or at risk of developing a secondary disease condition or side reaction, the treatment may be prophylactic.
(83) Treatment may be effected with the immunogenic composition or the vaccine as described herein, as the sole prophylactic or therapeutic agent or else in combination with any suitable means, e.g. including chemotherapy, radiotherapy or immunotherapy, such as passive antibody treatment against the tumor itself or indirectly against immune suppressive mechanism targeting PD-1 and/or CTLA4, as well as enzyme inhibitor therapy, e.g. checkpoint inhibitors such as AZD7762 or PF-477736.
(84) In cancer therapy, additional therapeutic treatments include, for instance, surgical resection, radiation therapy, chemotherapy, hormone therapy, anti-tumor vaccines, antibody based therapies, whole body irradiation, bone marrow transplantation, peripheral blood stem cell transplantation, and the administration of chemotherapeutic agents.
(85) The term “combination” as used in this regard, e.g. with respect to the combination of compounds or treatments specifically refers to the concomitant, simultaneous, parallel or consecutive treatment of a subject.
(86) For treatment the immunogenic composition or the vaccine as described herein may be administered at once, or may be divided into the individual components and/or a number of smaller doses to be administered at intervals of time. The vaccine is typically administered at a concentration of 0.1 to 500 μg/mL, e.g. either subcutaneously, intradermal, intramuscularly, intravenous, orally, through inhalation or intranasally, with or without an additional adjuvant such as ALUM. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data.
(87) The immunogenic composition or the vaccine as described herein can be administered by any suitable means and respective formulations for including, but not limited to, for example, any of the parenteral (including subcutaneous, intramuscular, intravenous and intradermal) injection, or local injection into the affected site, such as joints or into or around the tumor. In a preferred embodiment the vaccine is provided in a formulation for intramuscular, subcutaneous or intradermal injection.
(88) The invention also provides a delivery device, e.g. a syringe, pre-filled with the vaccine as described herein.
(89) Typically upon priming a subject by a first injection of a vaccine according to the invention, one or more booster injections may be performed over a period of time by the same or different administration routes. Where multiple injections are used, subsequent injections may be made, e.g. within 1 to 52 weeks of the previous injection, or even more.
(90) The vaccine typically may contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, as auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present among excipients. Typically, the vaccine according to the invention is prepared as an injectable, either as liquid solutions or suspensions, or solid forms suitable for solution in, or suspension in, liquid vehicles prior to administration. The preparations also may be emulsified or encapsulated in liposomes.
(91) Administration of the vaccine as described herein may be suitably and additionally be combined with any of the TLR9 agonists and/or further adjuvant measures, e.g. as separate entities in the same formulation or as separate formulations, to enhance the immune response.
(92) An enhanced Th1 immune response may include an increase in one or more of the cytokines associated with a Th1 immune response (such as IFNγ), and an increase in activated macrophages.
(93) An enhanced Th1 immune response may include one or more of an increase in antigen specific IgG antibodies, especially IgG1 antibodies.
(94) For example, the immunogenic composition or the vaccine as described herein, may be in association (e.g. chemically or recombinantly linked, bound by affinity binding or a mixture of separate components) with one or more adjuvants and/or pharmaceutically acceptable excipients. The vaccine may include one or more pharmaceutically acceptable excipients or vehicles, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Adjuvants may specifically be used to enhance the effectiveness of the vaccine. Adjuvants may be added directly to the vaccine compositions or can be administered separately, either concurrently with or shortly after, administration of the vaccine.
(95) Suitable adjuvants include cytokines and similar compounds which help orchestrate an immune response to the immunogen. As used herein, the term “cytokine” is used as a generic name for a diverse group of soluble proteins and peptides which act as humoral regulators at nano-to picomolar concentrations and which, either under normal or pathological conditions, modulate the functional activities of individual cells and tissues. These proteins also mediate interactions between cells directly and regulate processes taking place in the extracellular environment.
(96) Examples of cytokines include IL-1, IL-4, TNFα, IFNα, INFγ, GM-CSF, G-CSF
(97) CpG oligonucleotides can also be used as an adjuvant in conjunction with presentation of respective epitopes. Other adjuvants include alum, (in)complete Freund's adjuvant, B. pertussis or its toxin, IC31, etc.
(98) The components of the immunogenic composition, which are: the directed adjuvant component, e.g. the anti-CD32 moiety linked to the TLR9 ligand and the first peptidic alpha-helix; and the immunogen component, e.g. comprising the peptide immunogen linked to the second peptidic alpha-helix that matches the first one, as well as the immunogenic composition or the vaccine, or any of its binding moieties or ligands and the immunogen with our without the coil repeats may be obtained by various methods known in the art, e.g. by purification or isolation from cell culture, recombinant technology or by chemical synthesis.
(99) According to a specific embodiment, the immunogenic composition and/or the directed adjuvant component and/or the immunogen component thereof, is produced as a recombinant polypeptide, such as by recombinant DNA technology. As used herein, the term “recombinant” refers to a molecule or construct that does not naturally occur in a host cell. In some embodiments, recombinant nucleic acid molecules contain two or more naturally-occurring sequences that are linked together in a way that does not occur naturally. A recombinant protein refers to a protein that is encoded and/or expressed by a recombinant nucleic acid. In some embodiments, “recombinant cells” express genes that are not found in identical form within the native (i.e., non-recombinant) form of the cell and/or express native genes that are otherwise abnormally over-expressed, under-expressed, and/or not expressed at all due to deliberate human intervention. Recombinant cells contain at least one recombinant polynucleotide or polypeptide. “Recombination”, “recombining”, and generating a “recombined” nucleic acid generally encompass the assembly of at least two nucleic acid fragments. In certain embodiments, recombinant proteins and recombinant nucleic acids remain functional, i.e., retain their activity or exhibit an enhanced activity in the host cell.
(100) Thus, the invention further refers to the production of the immunogenic composition or the components thereof, and the recombinant means for such production, including a nucleic acid encoding the amino acid sequence, an expression cassette, a vector or plasmid comprising the nucleic acid encoding the amino acid sequence to be expressed, and a host cell comprising any such means. Suitable standard recombinant DNA techniques are known in the art and described inter alia in Sambrook et al., “Molecular Cloning: A Laboratory Manual” (1989), 2nd Edition (Cold Spring Harbor Laboratory press).
(101) Herein the term “subject” is understood to comprise human or mammalian subjects, including livestock animals, companion animals, and laboratory animals, in particular human beings, which are either patients suffering from a specific disease condition or healthy subjects.
(102) The invention further provides a kit of components for preparing the immunogenic composition of the invention, e.g. a pharmaceutical kit comprising one or more containers filled with the components. The kits can be used in the above-described methods. In a particular embodiment, the kit further comprises instructions for using the components of the immunogenic composition or the prepared immunogenic composition or vaccine of the invention.
(103) According to a specific example, the vaccine according to the invention comprises a recombinant polypeptide of the structure SEQ ID 9 or 10, as depicted in
(104) Similar polypeptides may comprise a leader sequence, the amino acid sequence of a specific anti-CD32 moiety, which is e.g. a recombinant scFv, a linker, a tag for purification purposes and the sequence of the peptidic alpha-helix pepE. This construct with or without the TLR9 ligand is also called “warhead”, which may then be used to construct a vaccine by combination with an immunogen linked to the counter alpha helix pepK.
(105) According to another specific example, the anti-CD32 moiety is an anti-CD32a peptide with the sequence of SEQ ID 37: ADGAWAWVWLTETAVGAAK.sup.24 used as an alternative to the ScFv.
(106) According to a further example, a stable coiled coil is established between the warhead scFv and the immunogen.
(107) It has proven that PBMC could be effectively stimulated with such immunogen or vaccine.
(108) In a further example it could be shown that the warhead mediated enhanced antigen presentation. T cells were effectively stimulated when the immunogen with the coil (the pepK coil) interacted with the warhead containing the counterpart coil (the pepE coil).
(109) In further examples treatment of non-human primates is described, using the vaccine of the invention, triggering effective B-cell response and T-cell response, in particular cytotoxic T-cell response. Such B-cell response and T-cell response is particularly reversible, thus, avoiding undesired autoimmune reactions.
(110) In a specific example, it has proven that antisera induced antibody response and ADCC activity.
(111) Therefore, the present invention provides for a unique immunogenic composition and vaccine, and respective applications.
(112) The foregoing description will be more fully understood with reference to the following examples. Such examples are, however, merely representative of methods of practicing one or more embodiments of the present invention and should not be read as limiting the scope of invention.
EXAMPLES
(113) In the following, the examples refer to the specific use of exemplary immunogenic compositions termed OQR200. If not indicated otherwise, the OQR200 as used herein comprises the Her2/neu domain of Cynomolgus monkeys origin (also termed OQR200m), specifically for use in the in vivo monkey studies. It is understood that human OQR200 (also termed OQR200h) is likewise produced and used, e.g. in the monkey model or in particular for human use.
Example 1: Materials
Example 1a: Amino Acid-Sequence CyErb2-Coil (SEQ ID 9; Her2/Neu from Cynomolgus Monkeys Including pepK of the S-TIR Technology)
(114) TABLE-US-00006 1 10 20 30 40 TQVCTGTDMK LRLPASPETH LDMLRHLYQG CQVVQGNLEL 50 51 60 70 80 TYLPTNASLS FLQDIQEVQG YVLIAHNQVR QVPLQRLRIV 90 100 101 110 120 RGTQLFEDNY ALAVLDNGNP LNNTTPVTGA SPGGLRELQL 130 140 150 151 160 RSLTEILKGG VLIQRNPQLC YQDTILWKDI FHKNNQLALT 170 180 190 200 LIDTNRSRAC HPCSPVCKGS RCWGESSEDC QSLTRIVCAG 201 210 220 230 240 GCARCKGPLP TDCCHEQCAA GCTGPKHSDC LACLHFNHSG 250 251 260 270 280 ICELHCPALV TYNTDTFESM PNPEGRYTFG ASCVTACPYN 290 300 301 310 320 YLSTDVGSCT LVCPLHNQEV TAEDGTQRCE KCSKPCARVC 330 340 350 351 360 YGLGMEHLRE VRAVTSANIQ EFAGCKKIFG SLAFLPESFD 370 380 390 400 GDPASNTAPL QPEQLRVFET LEEITGYLYI SAWPDSLPDL 401 410 420 430 440 SVLQNLQVIR GRILHNGAYS LTLQGLGISW LGLRSLRELG 450 451 460 470 480 SGLALIHHNT RLCFVHTVPW DQLFRNPHQA LLHTANRPED 490 500 501 510 520 ECVGEGLACH QLCARGHCWG PGPTQCVNCS QFLRGQECVE 530 540 550 551 560 ECRVLQGLPR EYVNARHCLP CHPECQPQNG SVTCFGPEAD 570 580 590 600 QCVACAHYKD PPFCVARCPS GVKPDLSYMP IWKFPDEEGT 601 610 620 630 640 CQSCPINCTH SCVDLDDKGC PAEQRASPLT GSGHHHHHHG 650 651 660 670 680 GGSGGKVSAL KEKVSALKEK VSALKEKVSA LKEKVSALKE
(115) The linker between Her2 and pepK (italics) containing the HIS tag is underlined (SEQ ID 4: GGGSHHHHHHGGGSGG) and may be replaced by any other linker e.g. GGGSGGGS (SEQ ID 5)
Example 1 b: Amino Acid-Sequence Erb2-Coil (SEQ ID 10; Her2/Neu from Humans Including pepK of the S-TIR Technology)
(116) TABLE-US-00007 1 10 20 30 40 TQVCTGTDMK LRLPASPETH LDMLRHLYQG CQVVQGNLEL 50 51 60 70 80 TYLPTNASLS FLQDIQEVQG YVLIAHNQVR QVPLQRLRIV 90 100 101 110 120 RGTQLFEDNY ALAVLDNGNP LNNTTPVTGA SPGGLRELQL 130 140 150 151 160 RSLTEILKGG VLIQRNPQLC YQDTILWKDI FHKNNQLALT 170 180 190 200 LIDTNRSRAC HPCSPVCKGS RCWGESSEDC QSLTRIVCAG 201 210 220 230 240 GCARCKGPLP TDCCHEQCAA GCTGPKHSDC LACLHFNHSG 250 251 260 270 280 ICELHCPALV TYNTDTFESM PNPEGRYTFG ASCVTACPYN 290 300 301 310 320 YLSTDVGSCT LVCPLHNQEV TAEDGTQRCE KCSKPCARVC 330 340 350 351 360 YGLGMEHLRE VRAVTSANIQ EFAGCKKIFG SLAFLPESFD 370 380 390 400 GDPASNTAPL QPEQLRVFET LEEITGYLYI SAWPDSLPDL 401 410 420 430 440 SVLQNLQVIR GRILHNGAYS LTLQGLGISW LGLRSLRELG 450 451 460 470 480 SGLALIHHNT RLCFVHTVPW DQLFRNPHQA LLHTANRPED 490 500 501 510 520 ECVGEGLACH QLCARGHCWG PGPTQCVNCS QFLRGQECVE 530 540 550 551 560 ECRVLQGLPR EYVNARHCLP CHPECQPQNG SVTCFGPEAD 570 580 590 600 QCVACAHYKD PPFCVARCPS GVKPDLSYMP IWKFPDEEGT 601 610 620 630 640 CQSCPINCTH SCVDLDDKGC PAEQRASPLT GSGHHHHHHG 650 651 660 670 680 GGSGGKVSAL KEKVSALKEK VSALKEKVSA LKEKVSALKE
(117) The linker between Her2 and pepK (italics) containing the HIS tag is underlined (SEQ ID 4: GGGSHHHHHHGGGSGG) and may be replaced by any other linker e.g. GGGSGGGS (SEQ ID 5)
Example 1c: Amino Acid-Sequence Erb2-Coil (SEQ ID 38; Her2/Neu from Humans Including pepK of the S-TIR Technology)
(118) TABLE-US-00008 1 10 20 30 40 TQVCTGTDMK LRLPASPETH LDMLRHLYQG CQVVQGNLEL 50 51 60 70 80 TYLPTNASLS FLQDIQEVQG YVLIAHNQVR QVPLQRLRIV 90 100 101 110 120 RGTQLFEDNY ALAVLDNGNP LNNTTPVTGA SPGGLRELQL 130 140 150 151 160 RSLTEILKGG VLIQRNPQLC YQDTILWKDI FHKNNQLALT 170 180 190 200 LIDTNRSRAC HPCSPVCKGS RCWGESSEDC QSLTRIVCAG 201 210 220 230 240 GCARCKGPLP TDCCHEQCAA GCTGPKHSDC LACLHFNHSG 250 251 260 270 280 ICELHCPALV TYNTDTFESM PNPEGRYTFG ASCVTACPYN 290 300 301 310 320 YLSTDVGSCT LVCPLHNQEV TAEDGTQRCE KCSKPCARVC 330 340 350 351 360 YGLGMEHLRE VRAVTSANIQ EFAGCKKIFG SLAFLPESFD 370 380 390 400 GDPASNTAPL QPEQLRVFET LEEITGYLYI SAWPDSLPDL 401 410 420 430 440 SVLQNLQVIR GRILHNGAYS LTLQGLGISW LGLRSLRELG 450 451 460 470 480 SGLALIHHNT RLCFVHTVPW DQLFRNPHQA LLHTANRPED 490 500 501 510 520 ECVGEGLACH QLCARGHCWG PGPTQCVNCS QFLRGQECVE 530 540 550 551 560 ECRVLQGLPR EYVNARHCLP CHPECQPQNG SVTCFGPEAD 570 580 590 600 QCVACAHYKD PPFCVARCPS GVKPDLSYMP IWKFPDEEGT 601 620 630 640 650 CQSCPINCTH SCVDLDDKGC PAEQRASPLT SSGGGSGGKV 660 661 670 780 SALKEKVSAL KEKVSALKEK VSALKEKVSA LKE
(119) The linker between Her2 and pepK (italics) does not contain the HIS tag and is underlined (SEQ ID 39: SSGGGSGG) and may be replaced by any other linker consisting of G and S amino acid residues of e.g. 5-30 amino acid length, such as GGGSGGGS (SEQ ID 5).
Example 1d: ScFV Warhead Containing Coil: Amino Acid Sequence of ScFV-Coil1 (SEQ ID 3)
(120) TABLE-US-00009 1 10 20 30 40 MELGLSWIFL LAILKGVQCE VQLQQSGPEL KKPGETVKIS 50 51 60 70 80 CKASGYTFTN YNWVKQAPGK GLKWMGWLNT YTGESIYPDD 90 100 101 110 120 FKGRFAFSSE TSASTAYLQI NNLKGMNEDM ATYFCARGDY 130 140 150 151 160 GYDDPLDYWG QGTSVIVSSG GGGSGGGGSG SGGGDIVMTQ 170 180 190 200 AAPSVPVTPG ESVSISCRSS KSLLHTNGNT YLHWFLQRPG 201 210 220 230 240 QSPQLLIYRM SVLASGVPDR FSGSGSGTAF TLSISRVEAE 250 251 260 270 280 DVGVFYCMQH LEYPLTFGAG TKLELKGSIS AWSHPQFEKG 290 300 301 310 PEVSALEKEV SALEKEVSAL EKEVSALEKE VSALEK
(121) AA 1-19: leader sequence (to secrete the product)
(122) IAA 20-271 sequence of ScFV (the VH domain is underlined, VI is double underlined) order of VH and VL domain may be swapped)
(123) IAA140-154 Linker may be changed to any linker used in ScFV preparation
(124) AA 272-279: StrepTag II for purification may be exchanged to any type of tag e.g. flag tag or HIS tag.
(125) AA280-281: short linker (may be longer)
(126) AA282-316: heptad repeat alpha helix (pepE) to form the coiled coil with the counter heptad repeat alpha helix in the immunogen (pepK). In the example 5 repeats are used, more repeats may cause auto-aggregation and less repeats will reduce the affinity, however 4 repeats are still functional. The minimal functional number of repeats for the coils used is 3 and 5
(127) A TRL9 agonist such as CpG may be coupled to the warhead using a chemical coupling e.g. using the Solulink antibody-oligo coupling KIT. A peptidic TRL9 agonist may be coupled using standard peptide chemistry. There are three classes of CpG, all may be used to prepare a warhead from a ScFV-coil1:
(128) TABLE-US-00010 CPG class A Group CpG-A: e.g. ODN2216: (SEQ ID 6) GGGGGACGATCGTCGGGGGG CpG class B Group CpG-B: e.g. ODN2006: (SEQ ID 7) TCGTCGTTTTGTCGTTTTGTCGTT CPG class C Group CpG-C e.g. ODNM362: (SEQ ID 8) TCGTCGTCGTTCGAACGACGTTGAT
Example 2: Method for Expression, Production and Purification of CyHerb2-Coil
(129) Cloning of Gene CyErbB2-His6-pepK on pTT5:
(130) The DNA coding the extracellular domain of the Cynomolgus epidermal growth factor receptor 2 (CyErbB2: accession number: EHH58073.1) fused to a His6 tag and pepK (see example 1a) was synthesized by GeneART (construct ID: 14AFGWKC).
(131) The final expression vector was obtained by cloning the synthesized gene in the pTT5 vector by digestion with EcoRI/BamHI (NEB#R0101/NEB#R0136, respectively) and standard molecular biology procedures. Shortly, 3.5 μg of pTT5 vector and 1 μg of GeneArt plasmid DNA (containing the gene) were digested with 20 U or 10 U of each enzyme in NEB3.1 buffer, respectively. The vector (approx. 4.4 kb) and fragment (approx. 2.0 kb) were separated by agarose gel and purified using Illustra Gfx PCR DNA and gel band purification kit (GE#28-9034-71) following kit instructions. Thereafter, vector (50 ng) and fragment (60 ng) were ligated using 200 U of T4 DNA ligase and the corresponding buffer (NEB#M020L). After an overnight incubation at 16° C., 5 μL of the ligation was transformed into 20 μL of NEB10beta chemically competent cells (NEB#C3019H). Six colony forming units (cfu) were used for inoculation of larger cultures and plasmid isolation using Qiaprep Spin Miniprep kit (Qiagen#27106). The correct cloning of the DNA containing the immunogen was first confirmed by digestion of plasmids with BamHI and NheI-HF (NEB#R0131) and then re-confirmed by DNA sequencing. Larger quantities of the DNA were prepared following manufactures' instructions of Qiagen Endofree plasmid Maxi kit#12381 or NucleoBond® Xtra Midi#740410.50.
(132) Transfection of HEK293-6E with Polyethylenimine:
(133) The expression vector harboring CyErbB2-His6-pepK gene was transfected in HEK293-6E suspension cells using linear Polyethylenimine (PEI). PEIpro™ (Polyplus#115-010) was mixed into the DNA solution in a ratio 1:2 (volume: mass). Commonly, 1 mg of DNA is used for transfecting 1 L of HEK293-6E cells at a density between 1.5-2×10{circumflex over ( )}6/mL in medium Freestyle F17 (Invitrogen#A13835-01) supplemented with 4 mM L-Glutamine (PAA#M11-004; 200 mM), Geneticin G418 50 μg/mL (Gibco#10131-027; 50 mg/mL) and Pluronic F-68 (Gibco#24040-032; 10%). After mixing, the complex PEI: DNA was added dropwise to 1 L flasks containing each 330 ml cell suspension in Freestyle-F17 media.
(134) Purification of CyErbB2-His6-pepK Protein:
(135) Five days post transfection, the cultures were harvested and cells pelleted by centrifugation at 6000 rpm for 30 min. The supernatant was further filtered through a Sartopore 2 150 membrane (sartorius#5441307H4-SS) with a cut off of 0.45+0.2 μm. Phenylmethanesulfonyl fluoride solution (PMSF, SIGMA#93482) was added to the filtrate to a final concentration of 0.1 mM to inhibit protease activity. Media was dialyzed against 1× phosphate buffer saline (PBS) (PBS 10×, Gibco#70013 or Lonza#BE17-5179) using a tangential flow filtration (TFF) device Millipore Labscale TFF system connected to three 10 kDa cut off TFF-cassettes (Merck-Millipore#PXC010C50). The filtrate was concentrated 50 times before the media was exchanged using at least 7 volumes of the buffer. The final concentrate was supplemented with imidazole 0-40 mM ((Merck#1.04716.0250) and a protease inhibitor cocktail (Complete, EDTA-free, Roche#05056489001). Upon removing particles by filtration over 0.45 μm pore size, the CyErbB2-His6-pepK (immunogen) was purified from the concentrate through the His tag. For that purpose, a HisTRAP HP column (GE#29-0510-21) was installed in a Äkta purifier system (GE). Sample was loaded at a flow rate of 0.5 ml/min which was not altered thereafter. Upon completion, PBS containing 30 mM imidazole was added without changing the flow rate till absorbance signal was dropping to a plateau. After a washing step with PBS-75 mM imidazole, the immunogen was eluted with PBS-300 mM imidazole. Fractions of 1 mL were collected and the ones containing the eluted peak were finally pooled and dialyzed in order to remove the imidazole.
Example 3: Method for Production of OQR200
(136) For the production of OQR200, Warhead (Example 1d) and CyErb2-coil (Example 1a) were mixed in molar ratio of 1:1 and incubated for 1 h on ice. Subsequently the solution was sterilized using an 0.22 μm milipore filter and finally adsorbed to Alum under constant rotation for 12 h at 4° C. OQR200 was stored at 4° C. until use.
(137) For the production of OQR200 for use in human subjects, the human Her2/neu antigen has been used. Warhead (Example 1d) and Erb2-coil (Her2/neu from humans including pepK of the S-TIR technology, Example 1c) were mixed in molar ratio of 1:1 and incubated for 1 h on ice. Subsequently the solution was sterilized using a 0.22 μm milipore filter and finally adsorbed to Alum under constant rotation for 12 h at 4° C. OQR200 (human Her2/neu) was stored at 4° C. until use.
Example 4: In Vivo Induction of Anti-Autologous Her2 Immune Responses
Example 4a: OQR200h
(138) Cynomolgus monkeys are immunized s.c. with 500 μl containing 167 μg OQR200h (formulated on Alum, produced as described in Example 3, however employing Erb2-coil (Example 1b) instead of the CyErb2-coil (Example 1a)) on d1, d15, d35 and boosted on d56 or d127. Blood samples for serum preparation are taken weekly always before immunization starting at d-7. Serum is stored at −200 C until use. PBMC are purified for T cell experiments of d1 and d79 (2 weeks after boost) and immediately tested for antigen specific proliferation.
Example 4b: OQR200m (Herein Also Referred to as OQR200)
(139) Cynomolgus monkeys were immunized s.c. with 500 μl containing 167 μg OQR200 (formulated on Alum, produced as described in Example 3), on d1, d15, d35 and boosted on d56 or d127. Blood samples for serum preparation were taken weekly always before immunization starting at d-7. Serum was stored at −200 C until use. PBMC are purified for T cell experiments of d1 and d79 (2 weeks after boost) and immediately tested for antigen specific proliferation.
Example 5: Detection of Antibody Response after Application of OQR200
(140) Method:
(141) 96 well plates (Maxisorp Thermo 442404) were coated with 1 μg/ml Her2-His less in PBS (10× diluted PBS Lonza 10×: BE17-517Q) at 4° C., overnight. The plates were washed twice with PBST (PBS+0.05% Tween 20, SIGMA P1379-500ML), followed by blocking for one hour with 3% BSA (fraction V Sigma A4919-25g) in PBST The whole assay was done at room temperature. After 3 times washing with PBST, the plates were incubated for 1 hour with 1% monkey serum in PBS. The plates were washed tree times and anti hlgG-AP 1:5000 (AbDSerotec 5211-8104) was incubated for 1 hour. After washing 3 times Streptavidin-HRP (GE RPN1231V) 1:5000 in PBST was added for 1 hour. The plates were washed 3 times and TMB (SIGMA T0440-1L) was added. After 15 min incubation, the reaction was stopped with H2504 30%, (Merck 1.00716.1000). The plates were read at 450-620 nm at Tecan infinite M2.
(142) Results:
(143) After immunization with OQR200m on d1, d15 and d36 (
Example 6: Biological Activity of Induced Antibody Response: ADCC
(144) Method:
(145) 5000 SKBR3 cells (CLS 300333) were seeded per well in a 96 well plate. Medium used for culturing was DMEM (Gibco 10938-025) with Pen Strep Glutamine 1:100 (Gibco 10378-016) and 10% FCS (Sigma F2442) and incubated for 22-24 h, 37° C., 5% CO2. Monkey sera were inactivated for 30 min on 56° C. After incubation an ADCC kit (Promega G7010) was used.
(146) In short: medium was replaced by 25 μl medium of the kit. 25 μl 10% heat inactivated monkey serum was added, followed by 25 μl effector cells. The wells were incubated for 6 hours at 37° C. and 5% CO2. After 15 minutes at room temperature, 75 μl Bio GLO Luciferase Assay reagent was added. After 20 min. the supernatant was transferred to 96 well white plate (Costar 3912). Luciferase activity as a measure for cell killing was measured with a Tecan infinite M2. Killing is shown relative to d1
(147) Results:
(148) Monkey sera from d28, d35, d42, d49, d63 and d70 were tested for the capacity to kill Her2 expressing SKBR3 cells. Sera were 1/100 diluted. Maximum killing was seen 2 weeks after 3.sup.rd immunization (
Example 7: Biological Activity of Induced Antibody Response: Competition with Trastuzumab (Herceptin) and Pertuzumab (Perjeta)
(149) Method:
(150) 96 well plates (Maxisorp Thermo 442404) were coated with 0.5 μg/ml Her2-His less in PBS (10× diluted PBS Lonza 10×: BE17-517Q) at 4° C., overnight. The plates were washed twice with PBST (PBS+0.05% Tween 20, SIGMA P1379-500ML), followed by blocking for one hour with 3% BSA (fraction V Sigma A4919-25g) in PBST. After 3 times washing with PBST, the plates were incubated for 30 minutes on ice with 20% monkey serum. 100 ng/ml Pertuzumab-biotin or Trastuzumab-biotin was added for 30 min. on ice. The plates were washed tree times and Streptavidin-HRP (GE RPN1231V) 1:5000 in PBST was added. After 1 hour incubation at Room temperature the plates were washed 3 times and TMB (SIGMA T0440-1L) was added. After 15 min incubation the reaction was stopped with H2S04 30%, (Merck 1.00716.1000).
(151) The plates were read at 450-620 nm at Tecan infinite M2. % inhibition was calculated by the following formula:
(152)
(153) Results:
(154) Animals were immunized with OQR200 on d1, d15, and d35. Sera taken between d50 and d88 were tested for their ability to block binding of Trastuzumab (
Example 8: Biological Activity of Induced Antibody Response: Proliferation Inhibition of Her2/Neu Expressing Tumor Cells
(155) Method:
(156) 5000 SKBR3 cells (CLS 300333) were seeded per well in a 96 well plate (Costar 3603). Culture medium (DMEM-CM) used for culturing was DMEM (Gibco 10938-025) with Pen Strep Glutamine 1:100 (Gibco 10378-016) and 10% FCS (Sigma F2442). The cells were incubated for 22-24 h, 37° C., 5% CO2.
(157) Monkey sera were inactivated for 30 min on 56° C. and diluted to a final concentration of 10% with DMEM-CM. The medium of the plates was replaced by the monkey sera samples and after 72 hours incubation at 37° C., 5% CO2 10% Alamar Blue (Invitrogen DAL1025) was added to the wells. After 3 hours fluorescence which corresponds to cell metabolic activity was measured at 560-590 nm. As positive control Trastuzumab (at optimal concentration of 10 μg/ml) and Pertuzumab (at optimal concentration of 10 μg/ml) were spiked in DMEM-CM containing 10% normal monkey serum.
(158) Results:
(159) Animals were immunized with OQR200 on d1, d15, and d35. Sera taken at day 64 were tested for their ability to inhibit proliferation of a Her/neu expressing breast cancer cell line (SKBR3) (
Example 9: Induction of T Cell Responses Against Autologous Her2
(160) Method:
(161) PBMCs (100.000/well) of OQR200 treated Cynomolgus monkeys were incubated for 3 days with 30 μg/ml CyErb2 (Her2/neu from cynomolgus monkeys) at 37° C./5% CO2. As a positive control PBMCs were incubated with 30 μg/ml ScFV (protein backbone of warhead present in OQR200).
(162) Proliferation was assayed by the incorporation of [3|H]-thymidine (0.5 Ci/well) during the final 18 hrs of a 2-day culture. Cells were harvested for β-scintillation counting (Topcount NXT, Packard, Ramsey, Minn., USA). T cells before immunization did not respond to either antigen but after immunization T cells responded to both, the ScFV and CyErb2, as indicated by an increase [3|H]-thymidine uptake after immunization.
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