Methods and compositions for immunotherapy of cancer

Methods and vaccines for inducing an immune response to a tumor associated antigen in a host are provided wherein the vaccine contains either a fusion protein of the tumor associated antigen fused to a truncated form of listeriolysin or a recombinant form of Listeria monocytogenes which grows and spreads and is capable of expressing the tumor associated antigen alone or as a listeriolysin fusion protein. Methods of suppressing formation of tumors and inhibiting growth of tumors in a host via administration of these vaccines are also provided.

BACKGROUND OF THE INVENTION

Stimulation of an immune response is dependent upon the presence of antigens recognized as foreign by the host immune system. The discovery of the existence of tumor associated antigens has now raised the possibility of using a host's immune system to intervene in tumor growth. Various mechanisms of harnessing both the humoral and cellular arms of the immune system are currently being explored for cancer immunotherapy.

Elements of the cellular immune response are capable of specifically recognizing and destroying tumor cells. The isolation of cytotoxic T cells (CTL) from tumor-infiltrating cell populations or from peripheral blood suggests that such cells play an important role in natural immune defenses against cancer (Cheever et al., Annals N.Y. Acad. Sci . 1993 690:101-112). CD8 T cells (TCD8 ) in particular, which recognize Class I molecules of the major histocompatibility complex (MHC)-bearing peptides of 8 to 10 residues derived from proteins located in the cytosols, are believed to play an important role in this response. There are now numerous examples of both mouse and human TCD8 that specifically recognize tumor cells and have therapeutic activity after adoptive transfer, in some cases inducing complete remission. However, despite the potential for T cells to eradicate tumors, it is obvious from the progressive growth of most cancers that many tumors escape recognition by TCD8 in vivo. The induction of sufficient T cells in vivo has been difficult. Though a variety of tumors have been found to be immunogenic, stimulation of an effective antitumor immune response has been difficult to demonstrate.

One explanation for this phenomena is that tumors may be capable of delivering antigen-specific signals to T cells, but not the co-stimulatory signals necessary for full activation of T cells. Co-stimulation of T cells occurs when a surface molecule, B7, on the presenting cells interacts with a T cell molecule known as CD28. It has been observed that T cells receiving the antigen-specific signal (but not B7) become unresponsive. Many tumor cells do not carry the B7 protein, therefore B7 has been added to cancer cells (Travis, J., Science 1993 259, 310-311). It has been demonstrated that expression of the co-stimulatory ligand B7 on melanoma cells induced the rejection of a murine melanoma in vivo (Townsend, S. E. and Allison, J. P., Science 1993, 259, 368-370). This rejection was found to be mediated by CD8 T cells; CD4 T cells were not required. These results suggest that B7 expression may render tumor cells capable of effective antigen presentation, resulting in their eradication in vivo.

The effects of localized secretion of cytokines on tumor progression has also been studied. Secretion of low levels of interleukin-2 (IL-2) in a mouse fibrosarcoma cell line transfected with the human IL-2 gene introduced via a retroviral vector was found to abrogate the tumorigenicity of these cells and induce a long lasting protective immune response against a subsequent challenge with a tumorigenic dose of parent cells (Gansbacher et al., J. Exp. Med . 1990, 172, 1217-1224). In another study, cells from a spontaneously arising murine renal cell tumor were engineered to -secrete large doses of interleukin-4 (IL-4) locally (Golumbek et al., Science 1991, 254, 713-716). Animals injected with the tumor cells rejected the IL-4-transfected tumors in a predominantly T cell-independent manner. However, these animals developed a T cell-dependent systemic immunity to the parental tumor. The systemic immunity was tumor-specific and mediated by CDB T cells. These experiments suggest that it may be possible to cure parental tumors by generating a systemic immune response by the injection of genetically engineered tumor cells.

There is also evidence to suggest that some tumor cells express low levels of class I molecules in vivo and in vitro. Intracellular antigens must be processed before presentation to CD8 T cells by major histocompatibility complex (MHC) class I molecules. The antigen processing efficiency of 26 different human tumor lines has been studied (Restifo et al., J. of Exp. Med . 1993, 177, 265-272). Three different cell lines, all human small cell lung carcinoma, consistently failed to process endogenously synthesized proteins for presentation to the T cells. Pulse chase experiments showed that MHC class I molecules were not transported by these cells lines from the endoplasmic reticulum to the cell surface. Northern blot analysis showed that these cells contained little or no mRNA encoding MHC-encoded proteosomes and transporter genes. Treatment with interferon enhanced expression of these mRNAs and reversed the observed functional and biochemical deficits. Thus, potential therapeutic applications which include enhancing antigen processing at the level of transcription of MHC-encoded proteosome and transporter genes was suggested.

Immunizing patients with recombinant BCG (bacille Calmette-Gu rin) or Salmonella bacteria carrying a gene coding for an antigenic peptide has also been suggested as an oral tumor immunotherapy (Boon et al. Annu. Rev. Immunol . 1994, 12, 337-65). Orally administered live attenuated Salmonella recombinant vaccine, which expressed the full length P. berghei circumsporozite antigen, has been shown to protect mice against malaria. This immune response was mediated by the induction of CD8 T cells (Aggarwal et al., J. of Exp. Med . 1990, 172, 1083-1090). It is suggested that live attenuated Salmonella recombinants may be useful in the study of other diseases where CTL-mediated immunity may be important. However, no other experiments were reported. BCG has also been implicated as a novel live-vaccine vehicle which may prove useful in stimulating both humoral and cellular immune response to a wide variety of viral, bacterial and protozoal antigens (Stover et al., Nature 1991 , 351 , 456 - 460 ).

It has now been found that the immune response to an antigen, and in particular a tumor associated antigen, can be induced by the administration of a vaccine comprising a listeriolysin fusion protein comprising a tumor associated antigen or a recombinant form of the intracellular bacterium Listeria monocytogenes which expresses a tumor associated antigen or fragment thereof. The recombinant form of Listeria monocytogenes can express the tumor associated antigen alone or as a listeriolysin fusion protein which comprises the tumor associated antigen. In one embodiment, one or more vectors comprising recombinant Listeria monocytogenes each expressing a different tumor associated antigen or fusion protein thereof, can be used in a vaccine to stimulate an immune response. In this embodiment, it is preferred that the expressed tumor associated antigens be fused to listeriolysin. In another embodiment, one or more fusion proteins, each fusion protein comprising a truncated form of listeriolysin fused to a different tumor associated antigen, can be used. As demonstrated herein, administration of the vaccines of the present invention decrease the size of existing tumors and inhibit formation of primary tumors. No other stimulation following antigen presentation was required to induce this response.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method of inducing an immune response to a tumor associated antigen in a host which comprises administering to a host having cancer an effective amount of a vaccine comprising a fusion protein of the tumor associated antigen fused to a truncated form of listeriolysin, or a recombinant form of Listeria monocytogenes which grows and spreads and is capable of expressing a tumor associated antigen. In this method, the recombinant form of Listeria monocytogenes can express the tumor associated antigen alone or as a listeriolysin fusion protein which comprises the tumor associated antigen.

Another object of the present invention is to provide fusion proteins which comprise tumor associated antigens fused to a truncated form of listeriolysin.

Another object of the present invention is to provide a vaccine for inducing an immune response to a tumor associated antigen which comprises a fusion protein of a tumor associated antigen fused to a truncated form of listeriolysin, or a recombinant form of Listeria monocytogenes which grows and spreads and is capable of expressing a tumor associated antigen. The recombinant form of Listeria monocytogenes used in these vaccines can express the tumor associated antigen alone or as a listeriolysin fusion protein which comprises the tumor associated antigen.

Another object of the present invention is to provide a method of suppressing formation of tumors in a host comprising administering to a host an effective amount of a vaccine comprising a fusion protein of a tumor associated antigen fused to a truncated form of listeriolysin, or a recombinant form of Listeria monocytogenes which grows and spreads and is capable of expressing a tumor associated antigen. In this method, the recombinant form of Listeria monocytogenes can express the tumor associated antigen alone or as a listeriolysin fusion protein which comprises the tumor associated antigen.

Yet another object of the present invention is to provide a method of inhibiting growth and/or killing tumor cells in a host comprising administering to a host an effective amount of this vaccine.

The following examples are provided for illustrative purposes only and are not intended to limit the invention.

EXAMPLES

A sequence encoding the first 420 amino acids of Listeriolysin O (LLO) and its promoter along with some upstream regulatory sequences was PCR amplified from L. monocytogenes chromosomal DNA (wild type strain 10403s) and ligated to PCR amplified DNA encoding NP, derived from plasmid pAPR502. (Young, J. F., U. Desselberger, P. Graves, P. Palese and A. Shatzman, Cloning and Expression of influenza virus genes , The Origin of Pandemic Influenza Viruses , W. G. Laver, eds., Elsevier, New York, 1983, p. 129). The construction resulted in an in-frame fusion plus the addition of two amino acids at the site of the fusion junction. The fusion was cloned into the shuttle plasmid pAM401, a shuttle vector able to replicate in both gram and gram bacteria which contains a gram chloramphenicol resistance gene and a gram chloramphenicol resistance gene (Wirth, R., F. Y. An and D. B. Clewell, J. Bacteriol . 1986, 165, 831). The resultant plasmid, pDP1659, was introduced into wild type L. monocytogenes (strain 10403s) by electroporation to yield L. monocytogenes strain DP-L1659. This recombinant strain was clearly able to make and secrete a fusion protein of the predicted size (105 kD) as determined by western blot analysis of the secreted proteins in the culture supernatants using anti-LLO polyclonal antiserum and anti-NP monoclonal antibody. The presence of the fusion gene under the control of the LLO promoter in a multicopy plasmid resulted in reduced secretion of the chromosomally encoded LLO, but not to the extent that it prevented escape of the bacteria from the vacuole or subsequent intracytoplasmic growth. However, this strain was not stable in the absence of chloramphenicol.

To construct L. monocytogenes strain, DP-L2028, which is stable in vivo and which was used in Examples 2 through 6, plasmid pDP-1659 was modified by inserting the prfA gene from 10403s and then used to transform a prfA- L. monocytogenes mutant DP-L1075. This resulted in L. monocytogenes strain DPL2028 which secretes the LLO-NP fusion protein stably in vivo and in vitro.

Treatment of Mice With LM-NP

One hundred and twenty Balb/c mice were divided into three groups of 40. One group was immunized with one-tenth of an LD50 of wild-type L. monocytogenes , one group was immunized with sterile saline and the third group was immunized with a recombinant L. monocytogenes vector transformed to secrete influenza nucleoprotein fusion protein(LM-NP). After two weeks, each group received a similar booster immunization. This immunization schedule was determined to produce strong CTL responses against influenza nucleoprotein. Two weeks after the last immunization, animals in each group were challenged subcutaneously with either CT26 or RENCA which had been transfected with the same influenza nucleoprotein gene that was used to transform the L. monocytogenes vector (CT26-NP or RENCA-NP, respectively) or with the parental CT26 or RENCA line. Each mouse was administered 5 10 5 tumor cells, which is 50 times the tumoricidal dose. Tumor growth was monitored every two days in these six groups of animals. Results from this study are shown in FIGS. 1 through 4 . The only group showing any protection from the tumoricidal dose was the animals which received the LM-NP fusion protein and which were challenged with the relevant tumor cell expressing NP. In the CT26-NP group, after 25 days, 6 of the animals showed no detectable tumor growth, 3 had tumors of less than 5.0 mm and one had a tumor of 9.0 mm. In the RENCA-NP group, none of the animals showed any signs of tumor growth. In contrast, all the mice in the other groups have tumors between 1.5 and 3.0 cm.

In order to maintain the foreign NP gene, CT26-NP is usually maintained on the antibiotic G418. It is believed that the small number of CT26-NP tumor cells that grew in the LM-NP immunized mice are cells which have lost the NP gene in the absence of G418.

CTL Generated by Immunizing Balb/c Mice With LM-NP can kill tumor cells CT26 and RENCA that express NP In Vitro

Mice were immunized with 0.1 LD 50 of LM-NP. Two weeks later, the mice were sacrificed and primary cultures were set up of spleen cells with either influenza infected (A/PR8/34) splenocytes ( FIG. 5A ) or with a synthetic peptide 147-158 known to represent the immunodominant epitope of the NP protein (FIG. 5 B). After four days in culture, the cytolytic activity of both populations was measured against CT26-NP, RENCA-NP and the parental cell lines CT26 and RENCA. A positive control was included (P815, a mastocytoma tumor cell line known to be efficiently lysed by H-2 d restricted CTL in the presence of the peptide or when infected by A/PR8/34). As FIG. 5A shows, RENCA-NP and CT26-NP, but not the parental lines, were lysed by NP specific effectors induced by immunizing with LM-NP and expanded with A/PR8/34. In FIG. 5B , a similar experiment in which the effectors were expanded with peptide show similar results.

Immunization by LM-NP Will Cause Elimination of RENCA Tumor Growth

In this experiment, immunization with LM-NP after tumor growth had been initiated caused regression and depletion of tumors. Tumor cells (5 10 5 ) were introduced subcutaneously to 30 mice. On Day 13, after measurable tumors (5 mm) had grown in the mice, they were divided into three groups of ten. Ten mice received LM-NP, 10 mice received wild type Listeria monocytogenes and ten received no further treatment. On Day 23 the mice were immunized again with either LM-NP or wild type Listeria monocytogenes . As FIG. 6 shows, only the mice that received the LM-NP fusion protein show regression of tumor growth to the point where the tumor was no longer visible in 9 out of 10 mice.

Immunization by LM-NP Will Cause Cessation of CT26NP Tumor Growth

The experiment described in Example 4 was also done with the colorectal CT26-NP tumor cells. CT26-NP is a much faster growing tumor and is also more unstable in its expression of NP. Nevertheless, in this experiment, it was also found that immunization by LM-NP after tumor growth has been initiated halts tumor growth. Tumor cells (5 10 5 ) were introduced subcutaneously to 30 mice. On day 10, after measurable tumors (5 mm) had grown in the mice, they were divided into three groups of ten. Ten mice received LM-NP, 10 mice received wild type Listeria monocytogenes , and 10 mice received no further treatment. On day 17 the mice were immunized again with either LM-NP or wild type Listeria monocytogenes . As FIG. 7 shows, only the mice that received the LM-NP fusion protein show a change in tumor growth. However, unlike the case with RENCA, regression of growth was not observed in as many mice. This may be because by Day 17, instability of the CT26-NP tumor cells resulted in many of the tumor cells losing the NP antigen.

Inhibition of Tumor Growth is Caused by CD8 T Cells

In this experiment, 30 mice were immunized with LM-NP using the same protocol as discussed in Example 2. Ten days after the last immunization, 10 mice were depleted of CD8 cells by immunizing with antibody 2.43 (specific for the CD8 molecule); 10 mice were depleted of CD4 cells by immunizing with GK 1.5 (specific for the CD4 molecule); and 10 mice were left with a complete T cell repertoire. (The protocol for depletion of CD8 or CD4 T cells was that as described by A. Kruisbeek, Current Protocols In Immunology , Coligan et al., eds, John Wiley & Sons, Inc., 1994, V.1, 4.1.1-4.1.2). After T cell depletion, the mice were challenged subcutaneously with 5 10 5 CT26-NP cells per mouse. As a control, 10 naive mice were also challenged with the same dose. As FIG. 8 shows, the group of mice in which the CD8 T cell subset was depleted showed similar tumor growth to the control (naive) group of mice. The mice in which the CD4 T cell subset was depleted showed reduced protection against tumor growth, indicating that CD4 cells play an accessory response in the control of tumor growth; and the mice with a complete T cell repertoire show protection against tumor growth induced by the LM-NP fusion protein.