# Patent

## Title
Fusion of non-hemolytic, truncated form of listeriolysin O to antigens to enhance immunogenicity

## Abstract

Methods for enhancing the immunogenicity of an antigen via fusion to a non-hemolytic truncated form of listeriolysin O are provided.

## Background
This invention was supported in part by funds from the U.S. government (NIH Grant No. CA69632) and the U.S. government may therefore have certain rights in the invention.


BACKGROUND OF THE INVENTION
Stimulation of an immune response is dependent upon the presence of antigens recognized as foreign by the host immune system. Bacterial antigens such as Salmonella enterica and Mycobacterium bovis BCG remain in the phagosome and stimulate CD4 T-cells via antigen presentation through major histocompatibility class II molecules. In contrast, bacterial antigens such as Listeria monocytogenes exit the phagosome into the cytoplasm. The phagolysosomal escape of L. monocytogenes is a unique mechanism which facilitates major histocompatibility class I antigen presentation of listerial antigens. This escape is dependent upon the pore-forming sulfhydryl-activated cytolysin, listeriolysin O (LLO).
The ability of L. monocytogenes to break down the vacuole within a host cell and enter the cytoplasm has led to its use as a recombinant vaccine. U.S. Pat. No. 5,830,702 describes vaccines comprising attenuated mutants of Listeria spp. genetically engineered to express foreign antigens in the cytoplasm of infected macrophages and other cells. Several approaches for expressing the antigen in Listeria spp. are described including generation of a fusion protein of a selected foreign antigen and a listerial protein, preferably an enzyme involved in lysis of host vacuoles. In particular, a fusion protein encoding the hly promoter and the first 416 amino acids of LLO fused in-frame to the entire coding sequence of the NP antigen was constructed in E. coli and on transformation to Listeria monocytogenes is demonstrated to secrete a 105 kDA protein that reacts with antiserum to LLO and NP (col. 24 of '702 patent). Recombinant L. monocytogenes secreting a fusion protein comprising listeriolysin O and NP (LLO-NP) was demonstrated to target infected cells for lysis by NP-specific class I-restricted cytotoxic T cells. In contrast, a hemolysin-negative L. monocytogenes strain expressing LLO-NP presented the antigen in a class II restricted manner (Ikonimidis et al. J. Exp. Med. 1994 180:2209-2218). Thus, from these studies it was surmized that hemolysin-dependent bacterial escape from the vacuole is necessary for class I presentation in vitro.
The escape function of L. monocytogenes has also been transferred to Bacillus subtilis and attenuated Salmonella ssp. strains (Bielecki, J. et al. Nature 1990 345:175-176, Gentschev et al. Infect. Immun. 1995 63:4202-4205). S. enteric and M. bovis BCG vaccine carriers which secrete listeriolysin O have also been constructed (Kaufmann, S. H. and Hess, J. Immunol. Lett. January 1999 65(1-2):81-4). These constructs are taught to be capable of introducing antigens into the MHC class II and MHC class I pathway, resulting in stimulation of both CD4 and CD8 T-cells. Comparison of S. enterica vaccines which display the same listerial antigen in secreted and somatic form showed the secreted antigen display to be superior to the somatic antigen display (Kaufman, S. H. and Hess, J. Immunol. Lett. January 1999 65(1-2):81-4).
WO 99/10496 discloses recombinant BCG strains secreting hemolytically active hly with an improved MHC class I-restricted immune response for use as a vaccine against tuberculosis.
Administration of purified listeriolysin O encapsulated in liposomes has also been reported to be effective in the induction of antigen-specific Th1-dependent protective immunity to various kinds of intracellular parasitic bacteria in vivo (Tanabe et al. Infect. Immun. February 1999 67(2):568-75).
It has now been found that the immune response to an antigen can be enhanced by fusion of the antigen to a non-hemolytic truncated form of listeriolysin O (ΔLLO).
SUMMARY OF THE INVENTION
An object of the present invention is to provide a method for enhancing the immunogenicity of an antigen which comprises fusing to the antigen a non-hemolytic truncated form of listeriolysin O (ΔLLO).



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram of an HPV-E7 chromosomal expression system constructed by integration of an E7 gene into the Listeria chromosome.
FIG. 2 is a diagram of a preferred multi-copy plasmid containing prfA and E7 fused to a truncated form of the hly gene (Δhyl) that produced ΔLLO.
FIG. 3 is a graph showing tumor immunotherapeutic efficacy of antigens expressed in L. monocytogenes. Tumor size in millimeters in mice is shown at 7, 14, 21, 28 and 56 days post tumor-inoculation. Naive mice are depicted by an open-circle; mice administered Lm-LLO-E7 are depicted by a filled circle; mice administered Lm-E7 are depicted by a square; mice administered Lm-Gag are depicted by an open diamond; and mice administered Lm-LLO-NP are depicted by a filled triangle.
FIG. 4 is a graph showing tumor immunotherapeutic efficacy of antigens expressed by Vaccinia. Tumor size in millimeters in mice is shown at 7, 14, 26, 34, 44, 55 and 65 days post tumor-inoculation. Naive mice are depicted by a filled triangle; mice administered Vac-LLO-E7 are depicted by an open circle; mice administered Vac-SigE7L are depicted by an X; and mice administered Vac-E7 are depicted by an open diamond.
FIG. 5 is a diagram of various Vaccinia virus constructs expressing different forms of HPV16 E7 protein.



DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method for enhancing the immunogenicity of a selected antigen by fusion of the selected antigen to a non-hemolytic truncated form of listeriolysin O. It has now been found that fusion of an antigen to a non-hemolytic truncated form of listeriolysin O results in an antigen with enhanced immunogenicity as compared to antigen alone. This truncated form of listeriolysin O fused to an antigen better enables cell mediated immunity and anti-tumor immunity as compared to antigen alone. Further, these fusion proteins need not be expressed by L. monocytogenes, but rather can be expressed and isolated from other vectors and cell systems routinely used for protein expression and isolation.
Listeriolysin O (LLO) binds to cholesterol-containing membranes wherein it oligomerizes to form pores. The oligomerization is dependent on the presence of a reduced cystine residue at position 484 in the sequence that is required for oligomerization. The hly gene encodes a proprotein of 529 residues (GenBank Accession No. P13128; SEQ ID NO:1), the first 25 amino acids are the signal sequence and are cleaved from LLO when it is secreted by the bacterium. Thus, the full length active LLO protein is approximately 504 residues. For purposes of the present invention, by “truncated form of LLO or ΔLLO” it is meant a fragment of LLO which does not contain the activation domain at the amino terminus including cystine 484. Thus, the ΔLLO used in the following experiments was 416 amino acids long (SEQ ID NO:2) as 88 residues from the amino terminus which is inclusive of the activation domain containing cystine 484. However, it is believed that other ΔLLOs without the activation domain, and in particular cystine 484, will also be effective.
Enhanced immunogenicity of an antigen following fusion to a non-hemolytic truncated form of listeriolysin O was demonstrated.
In a first set of experiments, the HPV-E7 antigen was expressed in L. monocytogenes. An L. monocytogenes recombinant that expressed E7 was made by chromosomal integration of the E7 gene under the control of the hly promoter and with the inclusion of the hly signal sequence to ensure secretion of the gene product. The site of integration into the chromosome by homologous recombination was into a region that is non-essential for Lm virulence. The scheme for this is depicted in FIG. 1. The advantage in using this type of transformation is that resulting recombinants are stably transformed and contain no drug selection markers since the CAT gene, included in the plasmid to select for successful transformants after electroporation, is excised during a second recombination event. The expression and secretion of the antigen from the resulting recombinants, Lm-E7, was verified by Western Blot. In addition, therapeutic effects of Lm-E7 were optimized. For example, it was found that the best results were achieved delivering the vaccine orally as compared to parenterally and in a combined protection and regression mode that requires priming with Lm-E7 before tumor challenge and then administering Lm-E7 therapeutically after tumor challenge. Table 1 provides more details for optimized anti-tumor effects observed in this model in three different tumor cell lines, TC-1, C3 and EL-4/E7. Bacteria were delivered orally 14 and 7 days prior to tumor challenge and days 7 and 14 following tumor challenge. Delivery of 106 bacteria intraperitoneally in a similar protocol provided no long-term protection. However, better protection was observed when Lm-E7 was delivered orally. More specifically, with this regimen approximately 50% of the animals remained tumor free in perpetuity and immunization seriously retarded tumor growth in all animals.







TABLE 1







Treatment with Lm-E7









Number of tumor free animals versus



total in study (number survived)











105 TC-1 on
106 C3 on
5 × 105 EL-4/E7


Treatment
day 60
day 42
on day 40





108 Lm-E7
3/8 (5)
4/8 (8)
4/8 (6)


108 Lm-Gag(ZY-18)
2/8 (2)
0/8 (0)
2/8 (0)


Naive
0/8 (0)
0/8 (0)
1/8 (0)









Animals administered TC-1 or EL-4/E7 tumor cells that were tumor free were re-challenged on day 60 with TC-1 or day 40 EL-4/E7, respectively. The two animals in each group that had been immunized with Lm-Gag grew tumors whereas the animals immunized with Lm-E7 remained tumor free until termination of the experiment (day 124 in the case of TC-1 and day 54 for EL-4/E7).

Compared to results previously disclosed with Lm-NP and the RENCA, CT-26 and B16F10-NP models (Pan et al. 1995), the Lm-E7 was less effective than expected. Accordingly, an Lm-E7 construct was prepared in accordance with the method taught for preparation of the Lm-NP construct of Pan et al. (Cancer Res. 1995 55:4776-4779).
Specifically, a second L. monocytogenes vaccine that expresses a E7 fusion protein, referred to as Lm-LLO-E7, was prepared by complementing a prfA-deletion mutant with a plasmid containing a copy of the prfA gene and a copy of the E7 gene fused to a form of the hly gene truncated to eliminate the hemolytic activity of the enzyme, ΔLLO (see FIG. 2). Functional LLO is maintained by the organism via the endogenous chromosomal copy of hly. The expression and secretion of the fusion protein was verified by Western blot.
The ability of the Lm-LLO-E7 and Lm-E7 vaccine to induce anti-tumor immunity was then compared in a regression model. As shown in Table 2, Lm-LLO-E7 was found to be more effective than Lm-E7.







TABLE 2







Number of mice cured of TC-1 tumor at conclusion of


experiment













Mice TC-1 free
Mice alive at
Mice alive



Treatment
at day 45
day 45
at day 134







Naive
0/8
0/8
0/8



Lm-LLO-E7
4/8
8/8
4/8



Lm-E7
0/8
7/8
0/8










Thus, expression of the foreign gene as a fusion protein with ΔLLO enhances the immunogenicity of the antigen.

Additional experiments were performed to compare the ability of Lm-E7 with Lm-LLO-E7 to induce the regression of established sub-cutaneous HPV-16 immortalized tumors from C57Bl/6 mice. Results from these experiments are depicted in FIG. 3. In these experiments, mice were immunized i.p. with 0.1 LD50 with one of four constructs, Lm-E7, Lm-Gag (isogenic with Lm-E7 except for the antigen expressed), Lm-LLO-E7 or Lm-LLO-NP. Lm-LLO-NP is isogenic with Lm-LLO-E7 but expresses influenza antigen. A second immunization was performed on day 14. As can be seen in FIG. 3, 6 of 8 mice immunized with Lm-LLO-E7 were cured of their tumors and remained tumor free. None of the other animals showed any regression of the established tumors. Similar results have been achieved for Lm-LLO-E7 under different immunization protocols. Further, just one immunization has been demonstrated to cure mice of established TC-1 of 5 mm diameter.
Additional experiments were performed to confirm the enhanced therapeutic efficacy of a fusion protein comprising the E7 antigen and a truncated form of listeriolysin O. In these experiments a vaccinia vector that expresses E7 as a fusion protein with a non-hemolytic truncated form of listeriolysin O was constructed. The WR strain of vaccinia was used as the recipient and the fusion gene was excised from the listerial plasmid and inserted into pSC11 under the control of the p75 promoter. This vector was chosen because it is the transfer vector used for the vaccinia constructs Vac-SigE7Lamp and Vac-E7 and would therefore allow direct comparison with Vac-LLO-E7. In this way all three vaccinia recombinants would be expressed under control of the same early/late compound promoter p7.5. In addition SC11 allows the selection of recombinant viral plagues to TK selection and β-galactosidase screening.
FIG. 5 shows the various vaccinia constructs used in these experiments. Vac-SigE7Lamp is a recombinant vaccinia virus that expressed the E7 protein fused between lysosomal associated membrane protein (LAMP-1) signal sequence and sequence from the cytoplasmic tail of LAMP-1 (Wu et al. Proc. Natl. Acad. Sci. USA 1995 92:11671-5; Lin et al. Cancer Res. 1996 56:21-6). It was designed to facilitate the targeting of the antigen to the MHC class II pathway.
The following modifications were made to allow expression of the gene product by vaccinia: (a) the T5XT sequence that prevents early transcription by vaccinia was removed from the 5′ portion of the LLO-E7 sequence by PCR; and (b) an additional XmaI restriction site was introduced by PCR to allow the final insertion of LLO-E7 into SC11. Successful introduction of these changes (without loss of the original sequence that encodes for LLO-E7) was verified by sequencing. The resultant pSC11-E7 construct was used to transfect the TK-ve cell line CV1 that had been infected with the wildtype vaccinia strain, WR. Cell lysates obtained from this co-infection/transfection step contain vaccinia recombinants that were plaque purified 3 times. Expression of the LLO-E7 fusion product by plaque purified vaccinia was verified by Western blot using an antibody directed against the LLO protein sequence. In addition, the ability of Vac-LLO-E7 to produce CD8+ T cells specific to LLO and E7 was determined using the LLO(91-99) and E7(49-57) epitopes of Balb/c and C57/BL6 mice, respectively. Results were confirmed in a chromium release assay.
Tumor rejection studies were performed with TC-1 following the same protocol as described supra. Two experiments were performed with differing delays before treatment was started. In one experiment, treatments were initiated when the tumors were about 3 mm in diameter (see FIG. 4). As of day 76, 50% of the Vac-LLO-E7 treated mice are tumor free and 25% of the Vac-SigE7Lamp mice are tumor free.
In the second experiment, TC-1 tumors were grown to a larger size (5 to 6 mm). The LLO-E7 fusion protein based vectors were then compared against a larger number of vectors. Although some of the vaccine groups showed significant temporary regression of TC-1, by day 65 the data clearly shows that Lm-LLO-E7 and Vac-LLO-E7 are the most effective vaccines with respect to the ability to permanently induce the regression of established TC-1. Only 12% of the Vac-SigE7Lamp treated mice were tumor free while 37% of the Vac-LLO-E7 and Lm-LLO-E7 mice were tumor free. All other mice were dead.
Thus, expression of the antigen as a fusion protein with a non-hemolytic truncated form of listeriolysin O in host cell systems in listeria and host cell systems other than listeria results in enhanced immunogenicity of the antigen. While comparative experiments were performed with vaccinia, a multitude of other plasmids and expression systems which can be used to express these fusion proteins are known. For example, bacterial vectors useful in the present invention include, but are not limited to Salmonella sp., Shigela sp., BCG, L. monocytogenes and S. gordonii. Viral vectors useful in the present invention include, but are not limited to, Vaccinia, Avipox, Adenovirus, AAV, Vaccinia virus NYVAC, Modified vaccinia strain Ankara (MVA), Semliki Forest virus, Venezuelan equine encephalitis virus, herpes viruses, and retroviruses. Naked DNA vectors can also be used.
Accordingly, the present invention provides methods for enhancing the immunogenicity of an antigen via fusion of the antigen to a non-hemolytic truncated form of listeriolysin O or ΔLLO. In a preferred embodiment, the fusion proteins are produced recombinantly via a plasmid which encodes the truncated form of the listeriolysin O and the antigen. However, the antigen may also be chemically conjugated to the truncated form of listeriolysin O. These fusion proteins can then be incorporated into vaccines for administration to animals, preferably humans, to invoke an enhanced immune response against the antigen of the fusion protein. As will be obvious to those of skill in the art upon this disclosure, such vaccines are particularly useful in the treatment of infectious and neoplastic diseases.
These vaccines may further comprise adjuvants. Examples of adjuvants useful in these vaccines include, but are not limited to, unmethylated CpG, quill glycosides, CFA, QS21, monophosphoryl lipid A, liposomes, and bacterial mitogens and toxins.
The following examples are provided for illustrative purposes only and are not intended to limit the invention.
EXAMPLES
Example 1
Tumor Cell Lines
TC-1 is a lung epithelial cell from C57BL/6 mice immortalized by HPV-16 E6 and E7 and transformed by pVEJB expressing activated human c-HA-ras. C3 is a mouse embryo cell frp, C57BL/6 mice immortalized with the complete genome of HPV16 and transformed with pEJ-ras. EL-4/E7 is the thymoma EL-4 retrovirally transduced with E7.
Example 2
Comparison of Efficacy of Lm-GG/E7, Lm-AZ/E7 and Vac-SigE7Lamp
TC-1 (1×105) or C-3 (5×105) tumor cells were implanted subcutaneously in mice and allowed to grow for 7 to 9 days by which time they were palpable ( ̃5 mm in size). Mice were then immunized i.p. with one of three constructs, Vac-SigE7Lamp (107 PFU), Lm-E7 (106 CFU) or Lm-LLO-E7 (107 CFU). Animals received Lm-LLO-E7 and LM-E7 on days 7 and 14. Surviving mice were re-challenged with 105 TC-1 on day 43.
Example 3
Comparison of Efficacy of Vac-LLO-E7, Vac-E7 and Vac-SigE7Lamp
Four groups of 8 mice were implanted with 105 cells of TC-1. After 7 days the tumors were approximately 4 mm in size. One group of mice was untreated. Each of the other groups received 107 PFU of either Vac-E7, Vac-LLO-E7 or Vac-Sig-E7-lamp 7. A booster dose was administered on day 14.
Example 4
Comparison of Efficacy of Vac-LLO-E7 and Lm-LLO-E7 with Various other Vectors
TC-1 tumor cells (2×105)were implanted s.c. on the left flank in 96 C57BL/6 mice and allowed to grow for 7 days. The mice were divided into groups of 8 mice and each group was treated with one of the following vaccine: naive (no vaccine); Vac SigE7Lamp, 107 PFU, i.p.; Vac-LLO-E7, 107 PFU, i.p.; or Lm-LLO-E7, 107 PFU, i.p. The animals received a booster immunization one week later. Tumor growth was followed every two days by caliper measurement and recorded as the average of the narrowest and longest surface length. Immune parameters were also determined.

## Claims

1. A method for enhancing the cell-mediated immunogenicity of a tumor antigen, wherein the cell-mediated immunogenicity comprises major histocompatability class I presentation of the tumor antigen, the method comprising fusing to the tumor antigen a non-hemolytic truncated form of listeriolysin O by recombinant expression of a plasmid encoding the truncated form of the listeriolysin O and the tumor antigen in a host cell other than listeria or by a nonlisteria vector system or by chemical conjugation of the tumor antigen to the truncated form of the listeriolysin O, wherein the amino acid sequence of the non-hemolytic truncated form of listeriolysin O consists the amino acid sequence set forth in SEQ ID NO:2.