阅读说明：本技术 Gpi锚定抗原的增强的免疫原性 (Enhanced immunogenicity of GPI-anchored antigens ) 是由 K·尼亚齐 W·塔德罗斯 A·辛 于 2019-01-15 设计创作，主要内容包括：本申请提出了通过修饰TAA的蛋白质部分以包括跨膜结构域和运输信号而允许针对GPI锚定肿瘤相关抗原的免疫应答增强的组合物和方法,所述运输信号将经修饰的蛋白质引导至内体或溶酶体区室。最优选地,所述经修饰的蛋白质将不再具有GPI锚或GPI连接序列。(The present application presents compositions and methods that allow for enhanced immune responses to GPI-anchored tumor-associated antigens by modifying the protein portion of the TAA to include a transmembrane domain and a trafficking signal that directs the modified protein to an endosomal or lysosomal compartment. Most preferably, the modified protein will no longer have a GPI-anchor or GPI-linking sequence.)

1. A recombinant hybrid protein comprising:

an antigenic moiety coupled to at least one transmembrane domain and a transport element;

wherein said antigenic moiety is at least a portion of a GPI-anchored protein; and is

Wherein the trafficking element directs the recombinant hybrid protein to a subcellular location selected from the group consisting of: circulating the endosomes, sorting the endosomes and lysosomes.

2. The hybrid protein of claim 1 wherein the GPI-anchor protein is CEA, PSCA, mesothelin or urokinase plasminogen activator receptor.

3. The hybrid protein of claim 1, wherein the GPI-anchor protein is a non-cancer disease-associated protein, optionally a variant surface protein of Trypanosoma brucei or a prion protein.

4. The hybrid protein of any one of the preceding claims, wherein the antigenic moiety lacks a functional GPI-anchor signal.

5. The hybrid protein of any one of the preceding claims, wherein the transmembrane domain comprises a T cell receptor, CD (e.g., CD α, CD β), CD134, CD137, CD154, KIRDS, OX, CD, LFA-1(CD11, CD), ICOS (CD278), 4-1BB (CD137), GITR, CD, BAFFR, HVEM (LITR), SLAMF, NKp (KLRF), CD160, CD, IL2 β, IL2 γ, IL7 α, ITGA, VLA, CD49, ITGA, IA, CD49, ITGA, VLGA, VLA-6, CD49, ITGAD, CD11, ITGAE, CD103, ITGAL, CD11, LFA-1, ITGAM, CD11, ITGAX, CD11, ITGB, CD160, ITGAX, CD229, CD-100, CD-100, TNFAM (CD-CD, ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR or at least a portion of the transmembrane domain of the alpha, beta or zeta chain of PAG/Cbp.

6. The hybrid protein of any one of the preceding claims, wherein the transmembrane domain is bound in-frame to the C-terminus of the antigenic moiety.

7. The hybrid protein of any one of the preceding claims, wherein the transmembrane domain is coupled to the C-terminus of the antigenic moiety through a peptide linker.

8. The hybrid protein of any one of the preceding claims, wherein the trafficking element comprises the endosomal trafficking element of CD1a, CD1c, or Lamp 1.

9. The hybrid protein of any one of the preceding claims, wherein the trafficking element is bound in-frame to the C-terminus of the transmembrane domain.

10. A recombinant nucleic acid comprising:

a sequence segment encoding the hybrid protein of any one of claims 1-9, operably linked to a promoter to drive expression of the hybrid protein.

11. The recombinant nucleic acid of claim 10, wherein the nucleic acid is a viral expression vector.

12. The recombinant nucleic acid of claim 10, wherein the viral expression vector is an adenoviral expression vector optionally deleted of the E1 and E2b genes.

13. The recombinant nucleic acid of any one of claims 10-12, wherein the promoter is a constitutive promoter or an inducible promoter.

14. The recombinant nucleic acid of claim 13, wherein the promoter is inducible by hypoxia, IFN- γ, or IL-8.

15. The recombinant nucleic acid of any one of claims 10-14, wherein the recombinant nucleic acid further comprises a sequence encoding at least one of a co-stimulatory molecule, an immunostimulatory cytokine, a protein that interferes with or down-regulates checkpoint inhibition, and an adjuvant polypeptide.

16. The recombinant nucleic acid of claim 15, wherein the co-stimulatory molecule is selected from the group consisting of: OX40L, 4-1BBL, CD80, CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, GITR-L, TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1 and LFA 3.

17. The recombinant nucleic acid of claim 15, wherein the immunostimulatory cytokine is selected from the group consisting of: IL-2, IL-12, IL-15 superagonists (ALT803), IL-21, IPS1 and LMP 1.

18. The recombinant nucleic acid of claim 15, wherein the protein that interferes is an antibody or antagonist to CTLA-4, PD-1, TIM1 receptor, 2B4, or CD 160.

19. The recombinant nucleic acid of claim 15, wherein the adjuvant polypeptide is calreticulin or a portion thereof having adjuvant activity, or wherein the adjuvant polypeptide is HMGB1 or a portion thereof having adjuvant activity.

20. A recombinant virus comprising the recombinant nucleic acid of any one of claims 10-19.

21. The recombinant virus of claim 20, wherein the recombinant virus is a replication-defective virus, and optionally wherein the recombinant virus is an adenovirus that lacks the E1 and E2b genes.

22. A recombinant antigen presenting cell comprising the recombinant nucleic acid of any one of claims 10-19 or expressing the hybrid protein of any one of claims 1-9.

23. A method of increasing the antigenicity of a GPI-anchored protein, said method comprising:

modifying the protein portion of the GPI-anchored protein to include at least one transmembrane domain and a transport element; and

wherein the trafficking element directs the modified protein to a subcellular location selected from the group consisting of: circulating the endosomes, sorting the endosomes and lysosomes.

24. The method of claim 23, wherein the GPI-anchored protein is CEA, PSCA, mesothelin, or urokinase plasminogen activator receptor.

25. The method of claim 23, wherein the GPI-anchored protein is a non-cancer disease-associated protein, optionally a variant surface protein of trypanosoma brucei or a prion protein.

26. The method of any one of claims 23-25, wherein said antigenic moiety lacks a functional GPI anchoring signal.

27. The method of any one of claims 23-26, wherein the transmembrane domain comprises a T cell receptor, CD (e.g., CD α, CD β), CD134, CD137, CD154, KIRDS, OX, CD, LFA-1(CD11, CD), ICOS (CD278), 4-1BB (CD137), GITR, CD, BAFFR, HVEM (LITR), SLAMF, NKp (KLRF), CD160, CD, IL2 β, IL2 γ, IL7 α, ITGA, VLA, CD49, ITGA, IA, CD49, ITGA, VLA-6, CD49, ITGAD, CD11, ITGAE, CD103, ITGAL, CD11, CD-1, ITGAM, CD11, ITGAX, CD11, ITGB, CD160, ITGAMA-160, ITGAMA, CD229, ITGAMA (CD) CD100, ITGAM, CD229, ITGAM, CD, ITGAB, CD2, CD229, CD, ITGAM, CD-6, CD-CD (CD-CD, ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR or at least a portion of the transmembrane domain of the alpha, beta or zeta chain of PAG/Cbp.

28. The method of any one of claims 23-27, wherein the transmembrane domain binds in-frame to the C-terminus of the antigenic moiety.

29. The method of any one of claims 23-28, wherein the transmembrane domain is coupled to the C-terminus of the antigenic moiety through a peptide linker.

30. The method of any one of claims 23-29, wherein the transport element comprises an endosomal transport element of CD1a, CD1c, or Lamp 1.

31. The method of any one of claims 23-30, wherein the transport element is bound in-frame to the C-terminus of the transmembrane domain.

32. A method of treating a tumor that expresses a GPI-anchored tumor-associated antigen, comprising:

administering a cell-based vaccine composition comprising a recombinant hybrid protein according to any one of claims 1-9, or administering a DNA or RNA-based vaccine composition comprising a recombinant nucleic acid according to any one of claims 10-19.

33. The method of claim 32, wherein the cell-based vaccine composition comprises a plurality of recombinant autologous cells of the patient.

34. The method of claim 33, wherein the recombinant autologous cells are antigen presenting cells.

35. The method of claim 32, wherein the cell-based vaccine composition comprises a plurality of recombinant yeast or bacterial cells.

36. The method of claim 32, wherein the DNA or RNA based vaccine composition comprises a recombinant adenovirus.

37. A pharmaceutical composition comprising the recombinant virus of claim 20 or the recombinant antigen presenting cell of claim 22.

38. The pharmaceutical composition of claim 37, formulated for infusion.

39. Use of the recombinant virus of any one of claims 20-21 in the treatment of cancer.

40. Use of the recombinant nucleic acid of any one of claims 10-19 in the preparation of a vaccine composition for the treatment of cancer.

Technical Field

Background

The background description includes information that may be useful in understanding the present invention. There is no admission that any information provided herein is prior art or relevant to the presently claimed invention, nor is it admitted that any publication specifically or implicitly referenced is prior art.

All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

Significant advances have been made in cancer immunotherapy that targets patient and tumor specific antigens (neo-epitopes). Although promising, identifying suitable antigens and subsequently generating a customized therapeutic composition is time consuming and expensive. Alternatively, immunotherapy may also target an antigen common to a particular tumor (i.e., a tumor-associated antigen (TAA)). However, not all TAAs are equally effective in different patient groups and several TAAs are often completely ineffective as immunogenic entities due to the Glycophosphatidylinositol (GPI) anchoring to the cell membrane, fig. 1 schematically illustrates the structure of the GPI anchor.

Unfortunately, various TAAs are GPI-anchored proteins, including, inter alia, CEA (carcinoembryonic antigen, commonly associated with epithelial cancers), PSCA (prostate stem cell antigen), mesothelin (commonly associated with mesothelioma, ovarian and pancreatic adenocarcinomas), and the urokinase plasminogen activator receptor (commonly associated with the growth and metastasis of aggressive cancers in many cancers, such as gastric cancer), and thus are not generally effective targets for cancer immunotherapy.

Although the membrane anchor can conceptually be removed from the protein, the modified protein is not transferred to the cell surface in most cases and thus does not function properly. On the other hand, certain phospholipid anchors of membrane-anchored glycoproteins may be replaced by transmembrane domains, such as the shown dictyostelium viscosum. However, such substitutions significantly reduce the residence time on the cell surface (CellBiol [ cell biology ] Vol.124, stages 1 and 2, 1 month 1994, 205-215), so that the modified protein is less likely to bind to the antibody.

Thus, even though various TAAs may conceptually serve as therapeutic targets common to many cancers, and thus may eliminate the need for personalized therapies, not all TAAs are sufficiently immunogenic. Accordingly, there is a need to provide improved compositions and methods that enhance the immunogenicity of TAAs, in particular GPI-anchored TAAs.

Disclosure of Invention

The subject matter of the present invention relates to various immunotherapeutic compositions and methods in which a GPI-anchored antigen is modified such that the modified antigen will no longer be coupled to a GPI moiety but instead include one or more transmembrane domains and cytoplasmic tail sequences derived or adapted from proteins that transport the antigen to endosomes or lysosome systems (e.g., CD1a, CD1c, Lamp1 moieties). Such modified antigens lead to increased stimulation of CD4+ (relative to unmodified GPI-anchored antigens) and also to increased stimulation of (multifunctional) CD8 and T cells.

In one aspect of the inventive subject matter, the inventors contemplate a recombinant hybrid protein comprising an antigenic moiety coupled to at least one transmembrane domain and a transport element. In a preferred aspect, the antigenic moiety is at least a portion of a GPI-anchored protein, and the trafficking element directs the recombinant hybrid protein to a subcellular location (typically a circulating endosome, sorting endosome, or lysosome).

For example, suitable GPI-anchored proteins are TAAs, including in particular CEA, PSCA, mesothelin and urokinase plasminogen activator receptors, and proteins associated with non-cancer diseases (e.g., variant surface proteins of trypanosoma brucei (trypanosoma cruzi) or prion proteins). In other examples, the antigenic moiety lacks a functional GPI-anchor signal, and/or the transmembrane domain comprises a T cell receptor, CD (e.g., CD α, CD β), CD134, CD137, CD154, KIRDS, OX, CD, LFA-1(CD11, CD), ICOS (CD278), 4-1BB (CD137), GITR, CD, BAFFR, HVEM (LIGHTR), SLAMF, NKp (KLRF), CD160, CD, IL2 β, IL2 γ, IL7 α, ITGA, VLA, CD49, ITGA, IA, CD49, ITGA, VLA-6, CD49, ITGAD, CD11, ITGAE, CD103, ITDNAX, CD11, LFA-1, ITGAM, ITGAX, CD11, CD229, ACAGB, CD-100, TNFAMG (CD), CD-100, TNTAMGB, CD-100, TNGG, TNTARG, ITGAX, CD-CD, ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR or at least a portion of the transmembrane domain of the alpha, beta or zeta chain of PAG/Cbp.

Although not limiting to the subject matter of the invention, the transmembrane domain is preferably in-frame with the C-terminus of the antigenic moiety (with or without a peptide linker) and most preferably comprises an endosomal transport element of CD1a, CD1C, or Lamp 1. Furthermore, it is generally preferred that the transport element is bound in-frame to the C-terminus of the transmembrane domain.

In another contemplated aspect of the inventive subject matter, the inventors also contemplate a recombinant nucleic acid comprising a sequence segment encoding a hybrid protein as contemplated herein operably linked to a promoter to drive expression of the hybrid protein. Most typically, the recombinant nucleic acid is a viral expression vector (e.g., an adenoviral expression vector, preferably lacking the E1 and E2b genes), and the promoter is a constitutive promoter or an inducible promoter (e.g., inducible by hypoxia, IFN-. gamma., or IL-8). In addition, contemplated recombinant nucleic acids may further comprise a sequence encoding at least one of a co-stimulatory molecule, an immunostimulatory cytokine, a protein that interferes with or down-regulates checkpoint inhibition, and an adjuvant polypeptide.

Suitable co-stimulatory molecules include OX40L, 4-1BBL, CD80, CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, GITR-L, TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1 and LFA3, while contemplated immunostimulatory cytokines include IL-2, IL-12, IL-15 superagonists (ALT803), IL-21, IPS1 and LMP1, and suitable interfering proteins include antibodies or antagonists against CTLA-4, PD-1, 1 receptors, 2B4 and/or CD 160. Contemplated adjuvant polypeptides are calreticulin or a portion thereof having adjuvant activity, or HMGB1 or a portion thereof having adjuvant activity.

Viewed from a different perspective, the inventors also contemplate recombinant viruses, particularly replication-defective viruses (e.g., adenoviruses lacking the E1 and E2b genes), comprising the recombinant nucleic acids set forth herein. Likewise, recombinant antigen presenting cells comprising the recombinant nucleic acids set forth herein are also contemplated.

In yet another aspect of the inventive subject matter, the inventors also contemplate a method of increasing the antigenicity of a GPI-anchored protein that includes the step of modifying the protein portion of the GPI-anchored protein to include at least one transmembrane domain and a trafficking element, wherein the trafficking element directs the modified protein to a subcellular location (e.g., circulating endosome, sorting endosome, or lysosome). With respect to GPI-anchor proteins, preferably the protein is a TAA (e.g., CEA, PSCA, mesothelin, and urokinase plasminogen activator receptor), or a protein associated with a non-cancer disease, optionally a variant surface protein of Trypanosoma brucei or a prion protein.

Accordingly, the inventors also contemplate a method of treating a tumor expressing a GPI-anchored tumor-associated antigen comprising the step of administering a cell-based vaccine composition comprising the contemplated recombinant hybrid protein, or a DNA or RNA-based vaccine comprising the contemplated recombinant nucleic acid composition.

For example, a suitable cell-based vaccine composition may comprise a plurality of recombinant autologous cells (preferably antigen presenting cells) of the patient, or may comprise recombinant yeast or bacterial cells. Similarly, DNA or RNA based vaccine compositions may comprise recombinant adenoviruses. Thus, contemplated pharmaceutical compositions may comprise a recombinant virus or recombinant antigen presenting cell as set forth herein (typically formulated for infusion). Viewed from a different perspective, the inventors also contemplate the use of the recombinant viruses presented herein in the treatment of cancer, and the use of the recombinant nucleic acids presented herein in the preparation of vaccine compositions for the treatment of cancer.

Various objects, features, aspects and advantages of the present subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawings in which like numerals represent like components.

Drawings

FIG. 1 is a schematic of an exemplary GPI-anchored protein.

Figure 2A is a schematic of an exemplary immunization protocol/schematic according to the inventive subject matter.

Figure 2B is an exemplary schematic diagram of a recombinant construct for modified CEA.

Fig. 3 shows exemplary results of CD4+ splenocytes following the immunization protocol of fig. 2A.

Fig. 4 shows exemplary results of CD8+ splenocytes following the immunization protocol of fig. 2A.

Figure 5 shows exemplary ELISA results following the immunization protocol of figure 2A.

Detailed Description

The inventors have now found that GPI-anchored membrane proteins, particularly disease-associated GPI-anchored membrane proteins, can be genetically modified to enhance their immunogenicity and make these antigens suitable targets for immunotherapy. Notably, by modifying the protein to enter the MHCII pathway, robust CD4+ cell activation can be achieved, thereby enabling cross presentation and supporting the growth of CD8+ cells through the CD4 "helper" function. This approach is particularly advantageous when the GPI-anchored membrane protein is a TAA, such as CEA, which is very common in epithelial cancers. Because of the GPI-anchor, CEA is not normally a therapeutically effective antigen that stimulates CD8T cells in its native configuration. However, the modification of the GPI-anchored membrane proteins proposed herein substantially increases immunogenicity, and thus may make GPI-anchored membrane proteins a therapeutic target.

For example, and as discussed in more detail below, by replacing the GPI anchor on CEA with a transmembrane domain and a cytoplasmic tail derived from proteins that enter the endosomal system (e.g., CD1a, CD1c, Lamp1), the inventors demonstrated that such modified proteins can increase CD4T cellular responses (e.g., assessed in terms of the frequency of antigen-specific IFN γ secreting cells and the frequency of TNF- α/IFN- γ secreting multifunctional T cells, which are ideal immune subtypes to combat cancer). Most notably, targeting CEA to the endosomal system using the modifications proposed herein also stimulated the number of IFN- γ secreting CD8T cells as well as the versatility of these cells. It is readily understood that these findings are associated with other TAAs, such as mesothelin, PSCA and urokinase plasminogen activator receptor, pathogen-encoded GPI-anchored proteins, and even such GPI-anchored proteins: as in autoimmunity, it is desirable to generate an immunosuppressive response to the protein by including immunosuppressive factors (e.g., IL-10, TGF-. beta.etc.). Other suitable GPI-anchor proteins and linking signals can be readily identified using Bioinformatics analysis (see, e.g., Bioinformatics 2005, Vol.21, No. 9, 2005, pp. 1846-1852; BMCBbioinformatics BMC Bioinformatics 2008, 9: 392), whereas known GPI-anchor proteins can be retrieved from various publicly accessible databases (e.g., URL: uniport. org).

Thus, the inventors contemplate generally the genetic modification of GPI-anchored proteins, in particular disease-associated GPI-anchored proteins, wherein the GPI-anchor is replaced by one or more transmembrane domains, and wherein the modified protein further comprises a trafficking element directing the recombinant hybrid protein so produced to a subcellular location favorable for MHC-II presentation, in particular a circulating endosome, a sorting endosome or a lysosome. Most typically, but not necessarily, the substitution of the GPI anchor to eliminate or reduce the efficiency of the GPI anchor modification can be achieved by removing the GPI anchor signal sequence or by modifying the GPI anchor signal sequence (e.g., substituting one or more amino acids to achieve the substitution of the GPI anchor). However, in less preferred aspects, GPI modification can also be accomplished by adding a peptide linker to the C-terminus of the protein followed by a transmembrane domain, or by adding a transmembrane domain to the C-terminus of the protein (here: no intervening linker is required).

With respect to transmembrane proteins contemplated, it will be appreciated that many domains are considered suitable herein, and that the modified protein may comprise one or more (e.g., two, three, four, six, etc.) transmembrane domains. For example, various receptor tyrosine kinases, cytokine receptors, receptor guanylate cyclases, and receptor serine/threonine protein kinases contain a single transmembrane domain. In other examples, certain ion channels and adenylate cyclases have six transmembrane domains, and selected cell surface receptors contain seven transmembrane domains. Exemplary transmembrane proteins include insulin receptor, insulin-like growth factor receptor, human growth hormone receptor, various glucose transporters, transferrin receptor, epidermal growth factor receptor, LDL receptor, leptin receptor, various interleukin receptors (e.g., IL-1 receptor, IL-2 receptor, etc.).

Most typically, contemplated transmembrane domains will comprise about 20 consecutive hydrophobic amino acids, which may be followed by charged amino acids. Many transmembrane domains are known in the art and all are considered suitable for use herein. For example, contemplated transmembrane domains may comprise a T cell receptor, CD (e.g., CD α, CD β), CD134, CD137, CD154, KIRDS, OX, CD, LFA-1(CD11, CD), ICOS (CD278), 4-1BB (CD137), GITR, CD, BAFFR, HVEM (LIGHT TR), SLAMF, NKp (KLRF), CD160, CD, IL2 β, IL2 γ, IL7 α, ITGA, VLA, CD49, ITGA, IA, CD49, ITGA, VLA-6, CD49, ITGAD, CD11, ITGAE, CD103, ITGAL, CD11, ITGAA-1, ITGAM, CD11, ITGAX, CD11, ITGB, LFGB, CD, TNFAA-1, ITGAE, CD160, ACAT 229, TAMGW, CD-150, SLAM (CD-100), SLMF-100, CD-100, ITGAM, ITGAX, ITGA, CD-6, ITGAE, ITGAD, CD-6, CD-6, CD-CD, SELPLG (CD162), LTBR or PAG/Cbp of alpha, beta or zeta chain of one or more membrane spanning region.

When multiple transmembrane domains are desired, it is noted that hybrid proteins may have at least two, three, four, or five or six transmembrane domains, or regions comprising at least about 10 to 35, more preferably about 15 to 30 or 20 to 25 amino acid residues, and having at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100% homology to known transmembrane domains. As will be readily appreciated, transmembrane domains can be identified by computational analysis of known amino acid sequences using predictive methods that analyze the secondary structure, hydrophobicity and/or topology of the associated protein (see, e.g., Biochemistry [ Biochemistry ] (1994)33: 3038-. Furthermore, in case the contemplated hybrid protein has multiple transmembrane domains, the individual transmembrane domains will typically be coupled to each other via an intracellular/extracellular loop, which may be formed of about 5 to 100 amino acids. Synthetic transmembrane domains may comprise repeating units of hydrophobic amino acids (e.g., F, W, V). Finally, it will be appreciated that while the use of a transmembrane domain is generally preferred, in at least some embodiments the transmembrane domain may also be omitted.

With respect to suitable transport elements, preferred elements contemplated include the CD1b leader sequence, the CD1a tail, the CD1c tail, and the LAMP1 transmembrane sequence. For example, as shown in more detail below, lysosomal targeting can be achieved using the LAMP1-TM (transmembrane) sequence; while circulating endosomes can be targeted via the CD1a tail targeting sequence; and sorting endosomes can be targeted via the CD1c tail targeting sequence.

In a particular embodiment, the immunogenic peptides of the invention further comprise an amino acid sequence (or another organic compound) that facilitates uptake of the peptide into (late) endosomes for processing and presentation within the CD1d determinant. Late endosomal targeting is mediated by signals present in the cytoplasmic tail of the protein and corresponds to recognized peptide motifs, such as a dileucine-based motif, a tyrosine-based motif or so-called acidic cluster motifs. The symbol o represents amino acid residues with bulky hydrophobic side chains, such as Phe, Tyr and Trp. Late endosomal targeting sequences allow processing and efficient presentation of antigen-derived T cell epitopes by the CD1d molecule. Such endosomal targeting sequences are comprised in, for example, the gp75 protein (JCellBiol [ J. Cell biol. ]130,807-820), the human CD 3. gamma. protein HLA-BM beta (J. Immunol. [ J. Immunol ] (1996)157,1017-1027), the cytoplasmic tail of the DEC205 receptor (JCellBiol [ J. Cell. Biol ] (2000)151, 673-683). Other examples of peptides that serve as sorting signals for the endosomes are disclosed in the Bonifacio and Traub reviews (annu. rev. biochem. [ annual review of biochemistry ] (2003)72, 395-447). See also Front Biosci [ bioscience frontier ] 2009.

Viewed from a different perspective, hybrid proteins contemplated herein include trafficking signals that will result in preferential trafficking of the hybrid protein to a desired subcellular location (e.g., at least 70%, more typically at least 80%, and most typically at least 90% of all expressed hybrid proteins are in the subcellular compartment of interest). Thus, in contemplated aspects of the inventive subject matter, the signal and/or leader peptide can be used to transport the neoepitope and/or polyepitope to an endosomal or lysosomal compartment. Thus, it should also be recognized that targeting pro-sequences and/or targeting peptides may be used. The pro sequence of the targeting peptide may be added to the N-terminus and/or C-terminus and typically comprises between 6-136 basic amino acids and hydrophobic amino acids. In the case of peroxisome targeting, the targeting sequence may be at the C-terminus. Other signals (e.g., signal spots) can be used and include sequence elements that are separated in the peptide sequence and function after appropriate peptide folding.

In addition, protein modifications such as glycosylation can induce targeting. Among other suitable targeting signals, the inventors contemplate peroxisome targeting signal 1(PTS1) (C-terminal tripeptide), and peroxisome targeting signal 2(PTS2) (nonapeptide located near the N-terminus). In addition, sorting of proteins into endosomes and lysosomes can also be mediated by signals within the cytosolic domain of the protein, which typically comprise short linear sequences. Some signals are referred to as tyrosine-based classification signals or dual leucine-based signals. All of these signals are recognized by components of the protein coat that are associated with the periphery of the cytosolic surface of the membrane. Other known signals are recognized by the Adaptor Protein (AP) complexes AP-1, AP-2, AP-3 and AP-4 as having characteristic specificities, while other signals are still recognized by another family of adaptor proteins called GGAs.

It will be further understood that in some embodiments, the transmembrane domain and transport sequence may be coupled to the antigen via a linker, which is preferably a flexible linker comprising 5 to 50 amino acids. For example, contemplated linkers include flexible glycine/serine linkers and rigid linkers. A variety of linker sequences are known in the art (see, e.g., Adv Drug Deliv Rev. [ advanced Drug delivery review ]2013, 10, 15; 65 (10): 1357-.

In further contemplated embodiments, the recombinant hybrid protein may also be modified to facilitate transport or retention to the cytoplasmic compartment (which may not necessarily require one or more specific sequence elements). For example, in at least some aspects, N-or C-terminal cytoplasmic retention signals can be added, including cytoplasmic retention signals of SNAP-25, syntaxin, synaptotagmin, synaptomains, vesicle-associated membrane proteins (VAMP), synaptobrevin (SV2), high affinity choline transporters, neurotoxins, voltage-gated calcium channels, acetylcholinesterase, and NOTCH. Thus, it will be appreciated that peptides can be routed to specific cellular compartments for preferential or even specific presentation by MHC-I or MHC-II.

The increased antigenicity of the recombinant hybrid proteins proposed herein can also be achieved by increasing one or more recombinant ubiquitination motifs (mono/poly) ubiquitination. There are many motifs known in the art, and all of these motifs are believed to be suitable for use herein (see, e.g., Proteins, 1/2 2010; 78 (2): 365-.

Although not limiting to the subject matter of the invention, the modified protein will generally be expressed in vitro or in vivo from appropriately constructed recombinant nucleic acids, and particularly suitable recombinant nucleic acids include plasmid and viral nucleic acids. In the case of viral nucleic acids, it is particularly preferred to deliver the nucleic acid by viral infection of the patient or patient cells. Thus, contemplated compositions may be administered as recombinant viral, yeast or bacterial vaccines, or as a mixture of multiple (usually different) proteins or hybrid polypeptides. Among other contemplated viral expression vectors and viruses, adenoviral vectors and viruses are specifically contemplated (e.g., AdV deleted from E2 b).

Viewed from a different perspective, it is understood that the compositions and methods presented herein will deliver poorly immunogenic antigens in a manner that promotes MHC-II presentation. Indeed, such modified proteins may be advantageously tailored to achieve a variety of specific immune responses, including enhanced CD4 ⁺Immune response and surprisingly enhanced CD8⁺An immune response. In addition, contemplated hybrid proteins may be co-expressed or co-administered with other immunostimulatory compositions (which may preferably be encoded on the same recombinant nucleic acid). For example, recombinant nucleic acids can be constructed that include expression cassettes encoding one or more of co-stimulatory molecules, immunostimulatory cytokines, and proteins that interfere with or down-regulate checkpoint inhibition. Suitable co-stimulatory molecules include OX40L, 4-1BBL, CD80, CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, GITR-L, TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1 and LFA3, and suitable immunostimulatory cytokines include IL-2, IL-12, IL-15 superagonists (ALT803), IL-21, IPS1 and LMP 1. In further contemplated aspects, preferred proteins that interfere with checkpoint inhibition include antibodies or antagonists to CTLA-4, PD-1, TIM1 receptor, 2B4, or CD 160. Similarly, the additional encoded signal comprises a protein adjuvant, such as calreticulin or HMBG protein (or fragment thereof)

Thus, in exemplary preferred aspects of the inventive subject matter, cancer immunotherapy may employ a recombinant adenovirus having a payload in which the modified TAA has a deleted or inoperative GPI anchor sequence, and further comprising a transmembrane domain and a trafficking signal as described above. Regardless of the type of recombinant virus, it is contemplated that the virus can be used to infect patient (or non-patient) cells ex vivo or in vivo. For example, the virus may be injected subcutaneously or intravenously, or may be administered intranasally or by inhalation to infect the patient's cells (and in particular antigen presenting cells). Alternatively, immune competent cells (e.g., NK cells, T cells, macrophages, dendritic cells, etc.) of a patient (or from an allogeneic source) can be infected in vivo and then delivered to the patient. Alternatively, immunotherapy need not rely on viruses, but may be effected by transfection with nucleic acids or vaccination using RNA or DNA, or other recombinant vectors that result in expression of the neoepitope (e.g., as a single peptide, in tandem minigenes, etc.) in the desired cells (and particularly in immune competent cells).

Most typically, the desired nucleic acid sequence (for expression from a virally infected cell) is under the control of suitable regulatory elements well known in the art. For example, suitable promoter elements include constitutively strong promoters (e.g., SV40, CMV, UBC, EF1A, PGK, CAGG promoters), but inducible promoters are also considered suitable for use herein, particularly under inducing conditions typical for a tumor microenvironment. For example, inducible promoters include promoters sensitive to hypoxia and promoters sensitive to TGF- β or IL-8 (e.g., via TRAF, JNK, Erk, or other responsive element promoters). In other examples, suitable inducible promoters include tetracycline-inducible promoters, myxovirus resistance 1(Mx1) promoters, and the like. Alternatively, it will be appreciated that the cancer vaccine composition need not be limited to adenoviral constructs as described above, but may include recombinant yeast and bacteria and recombinant proteins coupled to a carrier.

Where the expression construct is a viral expression construct (e.g., an adenovirus, and particularly AdV with deletions of E1 and E2 b), it is then contemplated that the recombinant virus may be used as a therapeutic vaccine, alone or in combination, in pharmaceutical compositions typically formulated as sterile injectable compositions, with a viral titer of 10 ⁶-10¹³Individual virus particles per dose unit, and more typically at 10⁹-10¹²Individual virus particles per dosage unit. Alternatively, the patient (or other HLA-matched) cells may be infected ex vivo with the virus and then the so-infected cells are delivered to the patient.In other examples, treatment of a patient with a virus may be accompanied by allogeneic or autologous natural killer or T cells in naked form or with a chimeric antigen receptor and expressing antibodies targeting a neoepitope, a tumor-associated antigen, or the same payload as the virus. Natural killer cells, including patient-derived NK-92 cell lines, may also express CD16 and may be conjugated to antibodies.

Additional therapeutic modalities based on neoepitopes (e.g., synthetic antibodies directed against neoepitopes as described in WO 2016/172722), alone or in combination with autologous or allogeneic NK cells, and in particular haNK cells or taNK cells (e.g., both commercially available from NantKwest, 9920 jackson great street carrefield City, 90232, california) may be employed, if desired. In the case of using haNK or taNK cells, it is particularly preferred that the haNK cells carry recombinant antibodies on CD16 variants that bind to the neo-epitope of the patient being treated, and in the case of using taNK cells, it is preferred that the chimeric antigen receptor of the taNK cells bind to the neo-epitope of the patient being treated. Additional modes of treatment may also be independent of neoepitopes, and particularly preferred modes include cell-based (e.g., activated NK cells (e.g., aNK cells, commercially available from NantKwest, 9920 jackson great street, cafe, 90232, california)) therapies; and non-cell based therapies (e.g., chemotherapy and/or radiation therapy). In still further contemplated aspects, the immunostimulatory cytokines (and in particular IL-2, IL15, and IL-21) may be administered alone or in combination with one or more checkpoint inhibitors (e.g., ipilimumab, nivolumab, etc.).

Similarly, it is still further contemplated that additional pharmaceutical intervention may comprise administration of one or more drugs that suppress immunosuppressive cells (and in particular MDSCs, tregs, and M2 macrophages). Thus, suitable drugs include inhibitors of IL-8 or interferon-gamma or antibodies that bind IL-8 or interferon-gamma; and agents that inactivate MDSCs (e.g., NO inhibitors, arginase inhibitors, ROS inhibitors); drugs that block cell development or differentiate into MDSCs (e.g., IL-12, VEGF inhibitors, bisphosphonates); or an agent that is toxic to MDSCs (e.g., gemcitabine, cisplatin, 5-FU). Also, drugs such as cyclophosphamide, daclizumab, and anti-GITR or anti-OX 40 antibodies can be used to inhibit Treg.

It is also contemplated that a low dose regimen may be used, preferably with chemotherapy and/or radiation delivered to the patient in a rhythmic manner, in order to trigger overexpression or transcription of the stress signal. For example, it is generally preferred that such treatment will employ a dose effective to affect at least one of protein expression, cell division and cell cycle, preferably to induce apoptosis or at least induce or increase expression of stress-related genes (and in particular NKG2D ligands). Thus, in further contemplated aspects, such treatment will include low dose treatment with one or more chemotherapeutic agents. Most typically, for chemotherapeutic agents, the exposure to low dose therapy will be LD ₅₀Or IC₅₀Equal to or less than 70%, equal to or less than 50%, equal to or less than 40%, equal to or less than 30%, equal to or less than 20%, equal to or less than 10%, or equal to or less than 5%. Additionally, in advantageous cases, such low dose regimens may be performed in a rhythmic manner as described, for example, in US7758891, US7771751, US7780984, US7981445, and US 8034375.

With respect to the particular drug used in such low dose regimens, it is contemplated that all chemotherapeutic agents are suitable. Kinase inhibitors, receptor agonists and antagonists, antimetabolites, cytostatics and cytotoxic drugs, among other suitable drugs, are contemplated herein. However, particularly preferred agents include those identified as interfering with or inhibiting components of the pathway that drives tumor growth or development. Analysis of the route to the chemical data, for example as described in WO 2011/139345 and WO 2013/062505, can be used to identify suitable drugs. Most notably, expression of the stress-related genes in the tumor cells thus obtained will result in surface presentation of NKG2D, NKP30, NKP44 and/or NKP46 ligands, which in turn activate NK cells to specifically destroy the tumor cells. Thus, it is understood that low dose chemotherapy can be used as a trigger in tumor cells to express and display stress-related proteins, which in turn will trigger NK cell activation and/or NK cell-mediated tumor cell killing. Additionally, NK cell mediated killing will be associated with the release of intracellular tumor specific antigens, which is thought to further enhance the immune response.

Examples of the invention

The inventors prepared various adenoviral expression constructs comprising an empty payload (group 1), a CEA payload (group 2), a CEA-CD1c payload (group 3), a CEA-LAMP1 payload (group 4), a CEA-CD1a payload (group 5). As shown in FIG. 2A, mice were immunized on a biweekly prime/boost regimen using 10 per injection¹⁰And (c) viral particles. All mice were euthanized on day 35 and splenocytes and peripheral blood were collected. Figure 2B schematically depicts a recombinant construct used in the scheme of figure 2A.

Fig. 3 provides exemplary results for CD4+ splenocytes. The left panel shows ICS stimulation of IFN γ observed in response to media (left), irrelevant peptide (SIVnef peptide, middle) and CEA peptide (right)⁺CD4⁺The proportion of cells. As expected, no media and no foreign protein pair of CD4 were found⁺The cells have obvious stimulation effect. However, all CEA-bearing adenovirus constructs produce significant responses when exposed to the CEA peptide, with the responses being greatly enhanced in the case of transport to the endo/lysosomal compartment. Also, IFN γ⁺TNFα⁺CD4⁺The proportion of cells increases significantly with trafficking to the endo/lysosomal compartment (right panel).

Even more noteworthy, when the same experiment was used to observe CD8 ⁺When the cells are cultured, the inventors found that IFN gamma⁺CD8⁺And IFN gamma⁺TNFα⁺CD8⁺In the case of cell trafficking to the intima/lysosomal compartment, the cells increased significantly, as can be seen in fig. 4. This enhancement is particularly pronounced relative to CEA peptide adenovirus delivery alone. Indeed, where the recombinant hybrid protein is targeted to the endosomal and/or lysosomal pathway (usually the MHC-I presentation pathway), reactive CD8⁺A significant increase in cells is particularly unexpected. Although not limited theretoThe subject of the present invention, however, is that it is contemplated that the recombinant hybrid proteins presented herein will advantageously undergo cross-presenting antigen processing. It will therefore be appreciated that the systems and methods considered not only greatly enhance the immune response against antigens that are otherwise difficult to target (GPI-anchored antigens), but also increase the proportion of multifunctional CD4+ and CD8+ cells. Advantageously, all immunized animals were also able to produce large amounts of antibodies, as shown in the graph depicting the anti-CEA ELISA in fig. 5.

In the above experiments the following sequences were used, where the leader peptide is shown underlined, the transmembrane domain is shown in bold, and the endosomal targeting sequence is shown in italics. Following the immunization protocol of fig. 2A, all sequences were subcloned and expressed from E2 b-deleted adenovirus AdV, which was subcutaneously injected into mice.

As used herein, the term "administering" a pharmaceutical composition or drug refers to direct and indirect administration of a pharmaceutical composition or drug, wherein direct administration of a pharmaceutical composition or drug is typically by a healthcare professional (e.g., physician, nurse, etc.), and wherein indirect administration includes providing the pharmaceutical composition or drug to the healthcare professional or making the pharmaceutical composition or drug available to the healthcare professional for direct administration (e.g., via injection, infusion, oral delivery, topical delivery, etc.). Most preferably, the recombinant virus is administered via subcutaneous or subdermal injection. However, in other contemplated aspects, administration may also be intravenous injection. Alternatively or additionally, antigen presenting cells may be isolated from or grown in cells of a patient, infected in vitro, and then delivered to the patient. Thus, it should be understood that contemplated systems and methods can be considered complete drug discovery systems (e.g., drug discovery, treatment regimens, verification, etc.) for highly personalized cancer treatment.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided with respect to certain embodiments herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. Accordingly, the inventive subject matter is not to be restricted except in light of the attached claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the claims recite at least one of something selected from the group consisting of A, B, C … … and N, the word should be construed to require only one element of the group, rather than A plus N, or B plus N, etc.