CA2253790C - Methods of enhancing anti-tumour immunity in a mammal - Google Patents
Methods of enhancing anti-tumour immunity in a mammal Download PDFInfo
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- CA2253790C CA2253790C CA002253790A CA2253790A CA2253790C CA 2253790 C CA2253790 C CA 2253790C CA 002253790 A CA002253790 A CA 002253790A CA 2253790 A CA2253790 A CA 2253790A CA 2253790 C CA2253790 C CA 2253790C
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Classifications
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- A61K2039/5152—Tumor cells
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- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55522—Cytokines; Lymphokines; Interferons
- A61K2039/55527—Interleukins
- A61K2039/55538—IL-12
Abstract
Anti-tumour immune responses are elicited by engineering genetically lymphoma tumour cells to express interleukin- 12 (IL- 12), the combination of B7-1 and interleukin- 12, or by increasing endogenous expression of the costimulatory molecule B7-2. The combination of B7-1 and IL-12 is most effective in minimal disease settings when the tumour cell engineered genetically to express both molecules is inoculated into the same site of tumour burden. In addition, increasing the expression of endogenous B7-2 provides another method by which anti-tumour immunity is generated against unmodified tumour cells. The mechanisms of the anti-tumour immune response are multifactorial involving T cells as well as natural killer (NK) cells.
Description
Methods of Enhancing Anti-Tumour Immunity in a Mammal Field of the Invention This invention relates to enhancement of anti-tumour immunity in a mammal when the genes for IL-12, B7-1 and B7-2 are introduced into a tumour cell either alone or in combinations. The invention further relates to methods of enhancing immunity in a mammal, as well as anti-tumour vaccines for use in a mammal.
Background of the Invention It has been possible to elicit an effective immune response in several murine tumour models by genetically manipulating tumour cells to express costimulatory molecules or cytokines. For example, transfection of the costimulatory molecule, B7-1, which is expressed on activated B and dendritic cells (Chen et al., 1992) has enabled certain murine tumour cells to be rejected in syngeneic hosts and allows for protection against subsequent challenge (Chen et al., 1992; Baskar et al., 1995; Townsend et al,1993). The introduction of cytokines may change the local immunological environment of tumour cells and may enhance the activation of tumour specific lymphocytes (Pardo11,1993;Ostrand-Rosenberg,1994). Interleukin-12 (IL- 12), for example, is produced by stimulated B cells and activated macrophages and has been shown to enhance the proliferation and cytolytic activity of T, NK and LAK cells in vitro (Kobayashi, et al., 1989; Robertson et al., 1992; Naume et al., 1992; Gately et al., 1991., 1992). Moreover, IL-12 promotes the production of IFN-y both in vitro (Gately et al., 1994) and in vivo (Chan et al., 1991). In several murine models, tumour cells genetically modified to express IL-12, or in vivo administration of IL-12 have shown anti-tumour effects (Brunda et al., 1993; Tahara et al., 1995; Tannenbaum et al., 1996;
Nastala et al., 1994; Zitvogel et al., 1995).
It has been shown that cooperative interactions between B7/CD28 and IL-12 allow for maximal proliferation and IFN-y production by a panel of Thl clones (Murphy et al., 1994). Also, it has been shown that B7-1 and IL-12 can augment specific lysis activity of C57BL/6 CD8+ T cells against the P1.HTR mastocytoma subclone in an allogeneic mixed lymphocyte-tumour culture even after depletion of CD4+ T cells (Gajewski et al., 1995).
Proliferation and cytokine production of peripheral blood human T cells has been induced in vitro with IL-12 and the addition of anti-CD28 antibodies (Kubin et al., 1994).
These results suggest that the combination of B 7-1 and IL- 12 may be important in eliciting anti-tumour immune responses in vivo.
This invention describes how anti-tumour immunity is augmented by introducing the genes for IL-12 and B7-1 into the same tumour cell. This strategy enables immunogenicity to be maximized while minimizing toxicity associated with high levels of systemically administered cytokines. The results illustrate that although B7-1 can increase the immunogenicity of the murine B-cell lymphoma cell line, A20, and delay tumour onset, only IL- 12 or B7-1 /IL-12 expressing variants elicit potent anti-tumour immune responses as demonstrated by both in vivo studies and in vitro cytotoxic T cell lysis (CTL) assays. Moreover, both CD4+ and CD8+ T cells appear to mediate this anti-tumour response. In minimal disease mixing experiment using the "Same Site" or "Different Site" methodology, the data show that although both B7-1/IL-12 and variants can increase survival at the different site, only the B7-1 /IL-12 variant can generate statistically improved surviva]_ At the same site, only the B7-1/inIL-12 var,iant can protect mice from tumour outgrowth.
Studies comparing the effectiveness of B7-1 and B7-2 in eliciting immunogenicity have yielded conflicting results. In some models,137-1 induees more potent anti-tumour responses than B7-2 (Qajewski et al., 1996; Matulonis et al., 1996). Because $7-1 is more effeetive at inducing Th1 than Th2 responses, the generation of CTLs may be involved in tumour rejection (Gajewski et al., 1996). In addition, data have suggested that the intrinsic immuraogenicity of the tumour may affect whetlier the expression of B7-t compared to B7-2 induces protective and curative ixxamunity (Cben, L. et al., 1994). In non-immunogenic adenocarcinoma or melanonia murine models, introduction of B7-provides superior protective imnnunity compared to B7-1 (Martin-Fontecha et al., 1996).
In contrast, immunogenic tumours engineered to express either 37-1 or B7-2 are equally ef,fective in establishing systemic anti-tumour immune responses (Martin-Fontecba et al., 1996). Similar enhancement of immunobenicity has also been demonstrated in Sal sarcoma cells transfected with either B7-1 or B7-2 (Baskar et al., 1995 ;Yang et al., 1995).
In most of the murine models to date, 87-1 or B7-2 has been introduced into tuznour cells which lack endogenous expression of either of these costimulatory molecules. It has not yet been determined whether tumours that endogenously express detectable levels of B7-1 or B7-2 can be made more irnmunogenic by furtller increasing this expression. This may be particularly relevant for non-Hodgkin's B cell iymphama where B7-2 expression has been documented in certain histologic subtypes. Schultze et al. have reported that in 6 out of 10 freshly isolated lymph node samples obtained from patients with follicular Iymphoma, low to int.emiediate levels of 137-2 and high levels of both MhYC I
and MHC
Background of the Invention It has been possible to elicit an effective immune response in several murine tumour models by genetically manipulating tumour cells to express costimulatory molecules or cytokines. For example, transfection of the costimulatory molecule, B7-1, which is expressed on activated B and dendritic cells (Chen et al., 1992) has enabled certain murine tumour cells to be rejected in syngeneic hosts and allows for protection against subsequent challenge (Chen et al., 1992; Baskar et al., 1995; Townsend et al,1993). The introduction of cytokines may change the local immunological environment of tumour cells and may enhance the activation of tumour specific lymphocytes (Pardo11,1993;Ostrand-Rosenberg,1994). Interleukin-12 (IL- 12), for example, is produced by stimulated B cells and activated macrophages and has been shown to enhance the proliferation and cytolytic activity of T, NK and LAK cells in vitro (Kobayashi, et al., 1989; Robertson et al., 1992; Naume et al., 1992; Gately et al., 1991., 1992). Moreover, IL-12 promotes the production of IFN-y both in vitro (Gately et al., 1994) and in vivo (Chan et al., 1991). In several murine models, tumour cells genetically modified to express IL-12, or in vivo administration of IL-12 have shown anti-tumour effects (Brunda et al., 1993; Tahara et al., 1995; Tannenbaum et al., 1996;
Nastala et al., 1994; Zitvogel et al., 1995).
It has been shown that cooperative interactions between B7/CD28 and IL-12 allow for maximal proliferation and IFN-y production by a panel of Thl clones (Murphy et al., 1994). Also, it has been shown that B7-1 and IL-12 can augment specific lysis activity of C57BL/6 CD8+ T cells against the P1.HTR mastocytoma subclone in an allogeneic mixed lymphocyte-tumour culture even after depletion of CD4+ T cells (Gajewski et al., 1995).
Proliferation and cytokine production of peripheral blood human T cells has been induced in vitro with IL-12 and the addition of anti-CD28 antibodies (Kubin et al., 1994).
These results suggest that the combination of B 7-1 and IL- 12 may be important in eliciting anti-tumour immune responses in vivo.
This invention describes how anti-tumour immunity is augmented by introducing the genes for IL-12 and B7-1 into the same tumour cell. This strategy enables immunogenicity to be maximized while minimizing toxicity associated with high levels of systemically administered cytokines. The results illustrate that although B7-1 can increase the immunogenicity of the murine B-cell lymphoma cell line, A20, and delay tumour onset, only IL- 12 or B7-1 /IL-12 expressing variants elicit potent anti-tumour immune responses as demonstrated by both in vivo studies and in vitro cytotoxic T cell lysis (CTL) assays. Moreover, both CD4+ and CD8+ T cells appear to mediate this anti-tumour response. In minimal disease mixing experiment using the "Same Site" or "Different Site" methodology, the data show that although both B7-1/IL-12 and variants can increase survival at the different site, only the B7-1 /IL-12 variant can generate statistically improved surviva]_ At the same site, only the B7-1/inIL-12 var,iant can protect mice from tumour outgrowth.
Studies comparing the effectiveness of B7-1 and B7-2 in eliciting immunogenicity have yielded conflicting results. In some models,137-1 induees more potent anti-tumour responses than B7-2 (Qajewski et al., 1996; Matulonis et al., 1996). Because $7-1 is more effeetive at inducing Th1 than Th2 responses, the generation of CTLs may be involved in tumour rejection (Gajewski et al., 1996). In addition, data have suggested that the intrinsic immuraogenicity of the tumour may affect whetlier the expression of B7-t compared to B7-2 induces protective and curative ixxamunity (Cben, L. et al., 1994). In non-immunogenic adenocarcinoma or melanonia murine models, introduction of B7-provides superior protective imnnunity compared to B7-1 (Martin-Fontecha et al., 1996).
In contrast, immunogenic tumours engineered to express either 37-1 or B7-2 are equally ef,fective in establishing systemic anti-tumour immune responses (Martin-Fontecba et al., 1996). Similar enhancement of immunobenicity has also been demonstrated in Sal sarcoma cells transfected with either B7-1 or B7-2 (Baskar et al., 1995 ;Yang et al., 1995).
In most of the murine models to date, 87-1 or B7-2 has been introduced into tuznour cells which lack endogenous expression of either of these costimulatory molecules. It has not yet been determined whether tumours that endogenously express detectable levels of B7-1 or B7-2 can be made more irnmunogenic by furtller increasing this expression. This may be particularly relevant for non-Hodgkin's B cell iymphama where B7-2 expression has been documented in certain histologic subtypes. Schultze et al. have reported that in 6 out of 10 freshly isolated lymph node samples obtained from patients with follicular Iymphoma, low to int.emiediate levels of 137-2 and high levels of both MhYC I
and MHC
II were expressed (Schultze et al., 1995). In addition, Van Gool et al., have shown that one out of four cases studied of follicle center cell lymphoma and two out of three cases of anaplastic large cell lymphoma expressed B7-2 (Van Gool et al., 1997).
Xerri et al., have also shown that the majority of follicular and diffuse large B cell lymphomas expressed both B7-1 (32/44 samples) and B7-2 (39/44 samples) (Xerri et al., 1997). In addition, it has been shown that expression of B7-1 and B7-2 was restricted to the malignant B cells of the germinal center (Dorfman et al., 1997). I liave shown expression of B7-2, but no expression of B7-1 in patient samples as well as B cell lines (data not shown).
Moreover, recent in vitro data demonstrate that low levels of B7 expression in human follicular lymphoma samples was unable to induce profound T cell proliferation, but was sufficient to prevent the induction of alloantigen-specific anergy. Activation of these malignant B cells by CD40 stimulation using CD40L transfectants upregulated B7-1 and B7-2 expression resulting in proliferation of allogeneic T cells (Dorfman et al., 1997). It was suggested that the quantitative level of B7 expression determines T cell responsiveness.
Because the A20 murine B cell lymphoma expresses low endogenous levels of B7-2, it was reasoned that this cell line would be ideal to investigate how modulation of levels of endogenous expression affects immunogenicity. A20 is non-immunogenic in that immunization with 106 irradiated cells or 105 live cells does not protect animals from challenge with live parental unmodified cells (Levitsky et al., 1996;
Pizzoferrato et al., 1997). This tumour cell line is tumourigenic despite moderate levels of B7-2 expression and high levels of both MHC I and II. Previous studies demonstrated that the immunogenicity of the A20 B cell lymphoma is enhanced with the introduction of J
and more potent responses are elicited with the combination of B7-1 and IL- 12 (Pizzoferrato et al., 1997).
In this study, the murine A20 cell line genetically engineered to express B7-2 at levels greater than the endogenous level elicit profound in vivo anti-tumour immunity. While animals injected with B7-1 variants succumb t.o tumour, animals inoculated with variants expressing greater levels of endogenous 137-2 reject tumour grow-th and are protected against subsequent challenge with unmodified parental cells. Moreover, CTLs are generated in protected animals. The potency of the anti-tumour response is equal to that observed with A20 variants genetically modified to express IL- 12 or both B7-1 and IL-12. In addition, vaccination of tumour bearing animals demonstrates the therapeutic potential of B7-2 in preventing tumour growth. Collectively, this murine model suggests that the level of B7-2 expression determines in vivo anti-tumour responsiveness. Note that B7-2 is already present on A20. Thus, these results imply that modestly increasing its level of expression is sufficient to change a non-immunogenic tumour into an immunogenic tumour.
5a In accordance with an aspect of the present invention, there is provided an in vitro or ex vivo method of enhancing immunity against hematopoietic tumours in a mammal which has a hematopoietic tumour, said method comprising the steps of:
genetically modifying hematopoietic tumour cells from the same or a different mammal to express a cytokine and costimulatory molecule, in the mammal, wherein said cytokine and costimulatory molecule is 1L-12 in combination with B7-l, and expressing said cytokine and costimulatory molecule in the presence of cells from said hematopoietic tumour, or, genetically modifying hematopoietic tumour cells from the same or a different mammal to express a costimulatory molecule, in the mammal, wherein said costimulatory molecule is B7-2, and expressing said costimulatory molecule in the presence of cells from said hematopoietic tumour.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the step of genetically modifying further comprises the step of genetically modifying said hematopoietic tumour cells using at least one vector construct which directs the expression of a DNA sequence, said at least one vector construct comprising:
IL-12 gene in combination with B7-2 gene; IL-12 gene in combination with B7-1 gene; B7-1 gene alone ; B7-2 gene alone; or IL-12 gene in combination with B7-1 gene in combination with B7-2 gene, to form genetically modified hematopoietic tumour cells, wherein said combination may be on one vector or on more than one vector.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the at least one vector construct comprises a polynucleotide vector.
5b In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the at least one vector construct comprises a combination of a virus and a plasmid; or a virus and a polynucleotide; or a plasmid and a polynucleotide; or a virus and a plasmid and a polynucleotide.
In accordance with another aspect of the present invention, there is provided the tnethod of the present invention wherein the at least one vector construct comprises a virus.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the virus is fowlpox.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the virus is canarypox.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the virus is adenovirus.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the virus is vaccinia virus.
In accordance with another aspect of the present invention, there is provided the method of the present invention whereiin the virus is swine pox virus or polio virus.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the virus is a retrovirus.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the at least one vector construct comprises a plasmid.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the plasmid is a DNA plasmid comprising a promoter which allows for the expression of the gene or combination of genes.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the plasmid is pRc/CMV.
5c In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the hematopoietic tumour is a leukemia, lymphoma, or myeloma.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the leukemia is acute lymphoblastic leukemia (ALL), acute myelocytic leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL) or juvenile myelo-monocytic leukemia (JMML).
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the lymphoma is B-Cell Burkitt's lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Ki-1 positive anaplastic large cell lymphoma, T cell lymphoma or histiocytic lymphoma.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the myeloma is a multiple myeloma.
In accordance with another aspect ofthe present invention, there is provided the method of the present invention wherein the genetically modified tumour cells are present at the same site as a tumour burden.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the genetically modified tumour cells are present at a site which is different frorii, a tumour burden.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the genetically modified tumour cells are present at a site which is distant from a tumour burden.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein said tumour burden is defined as a minimal disease state.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the genetically modified tumour cells are present in proximity to the hematopoietic tumour.
5d In accordance with another aspect of the present invention, there is provided an in vitro or ex vivo method of enhancing immunity against hematopoietic tumours in a mammal which has a hematopoietic tumour, said method comprising the steps of:
genetically modifying hematopoietic tumour cells from the same or a different mammal to express a cytokine and costimulatory molecule, in the mammal, wherein said cytokine and costimulatory molecule is IL-12 in combination with B7-1, and expressing said cytokine and costimulatory molecule, in vitro or ex vivo in the presence of immune cells, said immune cells comprising antigen presenting cells, T cells, and/or NK cells, in the presence of naturally occurring mammalian hematopoietic tumour cells, or genetically modifying hematopoietic tumour cells from the same or a different mammal to express a costimulatory molecule, in the mammal, wherein said costimulatory molecule is B7-2, and expressing said costimulatory molecule, in vitro or ex vivo, in the presence of immune cells, said immune cells comprising antigen presenting cells, T cells, and/or NK cells, in the presence of naturally occurring mammalian hema.topoietic tumour cells, and waiting until lysis or activation of cell death processes of said naturally occurring mammalian hematopoietic tumour cells occurs.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the expression of cytokine and costimulatory molecule in vitro or ex vivo comprises introducing at least one vector construct which directs the expression of a DNA sequence, said at least one vector construct comprising:
IL-12 gene in combination with B7-2 gene; IL-12 gene in combination with B7-1 gene; B7-l gene alone ; and 87-2 gene alone; or IL-12 gene in combination with B7-1 gene in combination with B7-2 gene, 5e into the vicinity of the hematopoietic tumour, wherein said combination may be on one vector or on more than one vector.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the step of genetically modifying further comprises the step of genetically modifying said hematopoietic tumour cells using at least one vector construct which directs the expression of a DNA sequence, said at least one vector construct comprising:
IL-12 gene in combination with B7-2 gene; IL-12 gene in combination with B7-1 gene; B7-1 gene alone ; B7-2 gene alone; or IL-12 gene in combination with B7-1 gene in combination with B7-2 gene, to form genetically modified hematopoietic tumour cells, wherein said combination may be on one vector or on more than one vector.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the at least one vector construct comprises a polynucleotide vector.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the at least one vector construct comprises a combination of a virus and a plasmid; or a virus and a polynucleotide; or a plasmid and a polynucleotide; or a virus and a plasmid and a polynucleotide.
In accordance with another aspect of the present invention, there is provided the nlethod of the present invention wherein the at least one vector construct comprises a virus.
In accordance with another aspect of the present invention, there is provided the niethod of the present invention wherein the virus is fowlpox.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the virus is canarypox.
5f In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the virus is adenovirus.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the virus is vaccinia virus.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the virus is swine pox or polio virus.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the virus is a retrovirus.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the at least one vector construct comprises a plasmid vector.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the plasmid is a DNA plasmid comprising a promoter which allows for the expression of the gene or combination of genes.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the plasmid is pRc/CMV.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the hematopoietic tumour is a leukemia, lymphoma, or myeloma.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the leukemia is acute lymphoblastic leukemia (ALL), acute myelocytic leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL) or juvenile myelo-monocytic leukemia (JMML).
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the lymphoma is B-Cell Burkitt's lymphoma, Hodgkin's lymphomas, non-Hodgkin's lymphomas, Ki-l positive anaplastic large cell lymphomas, T cell lymphomas or histiocytic lymphomas.
5g In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the myeloma is a multiple myeloma.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the genetically modified tumour cells are present at the same site as a tumour burden.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the genetically modified tumour cells are present at a site which is different from a tumour burden.
In accordance with another aspect of the present invention, there is provided the method of the present invention whereiin the genetically modified tumour cells are present at a site which is distant from a tumour burden.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein said tumour burden is defined as minimal disease state.
In accordance with another aspect of the present invention, there is provided a vaccine for enhancing immunity to hematopoietic tumours in a mammal which has a heniatopoietic tumour, said vaccine comprising genetically modified hematopoietic tumour cells of a mammal, wherein said tumour cells are genetically modified to express a cytokine and costimulatory molecule wherein said cytokine and costimulatory molecule is IL-12 in combination with B7-l, or wherein said tumour cells are genetically modified to express a costimulatory molecule wherein said costimulatory molecule is B7-2.
5h In accordance with another aspect of the present invention, there is provided the vaccine wherein the vaccine comprises live, replicating cells.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the vaccine comprises non-replicating cells.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the genetically modified hematopoietic tumour cells further comprise hematopoietic tumour cells transduced, transfected or infected using at least one vector construct which directs the expression of a DNA sequence, said at least one vector construct comprising:
IL-l2 gene in combination with B7-2 gene; IL-12 gene in combination with B7-1 gene; B7-I gene alone ; B7-2 gene alone; or IL-12 gene in combination with B7-l gene in combination with B7-2 gene, wherein said combination may be on one vector or on more than one vector.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the at least one vector construct comprises a polynucleotide vector.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the at least one vector construct comprises a combination of a virus and a plasmid; or a virus and a polynucleotide; or a plasmid and a polynucleotide; or a virus and a plasmid and a polynucleotide.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the at least one vector construct comprises a virus.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the virus is fowlpox.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the virus is canarypox.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the virus is adenovirus.
5i In accordance with another aspect of the present invention, there is provided the vaccine wherein the virus is vaccinia virus.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the virus is swine pox virus or polio virus.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the virus is a retrovirus.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the at least one vector construct comprises a plasmid.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the plasmid is a DNA plasmid comprising a promoter which allows for the expression of the gene or combination of genes.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the plasmid is pRc/CMV.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the hematopoietic turnour is a leukemia, lymphoma, or myeloma.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the leukemia is acute lymphoblastic leukemia (ALL), acute myelocytic leukemia (AML), chronic rnyeloid leukemia (CML), chronic lymphocytic leukemia (CLL) orjuvenile myelo-monocytic leukemia (JMML).
In accordance with another aspect of the present invention, there is provided the vaccine wherein the lymphoma is B-Cell Burkitt's lymphoma, Hodgkin's lymphomas, non-Hodgkin's lymphomas, Ki-1 posiltive anaplastic large cell lymphomas, T
cell lymphomas or histiocytic lymphomas.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the myeloma is a multiple myeloma.
In accordance with another aspect of the present invention, there is provided a pharmaceutical composition for use as a vaccine for enhancing immunity to hematopoietic tumours in a mammal which has a hematopoietic tumour, said 5j pharmaceutical composition comprising, genetically modified hematopoietic turnour cells of a mammal wherein said tumour cells are genetically modified to express a cytokine and costimulatory molecule wherein said cytokine and costimulatory molecule is IL-12 in combination with or genetically modified hematopoietic turnour cells of a mammal wherein said tumour cells are genetically modified to express a co-stimulatory molecule wherein said costimulatory molecule is B7-2;
together with a pharmaceutically acceptable carrier or diluent.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein said genetically modified hematopoietic tumour cells further comprise hematopoietic tumour cells transduced, transfected or infected using at least one vector const:ruct which directs the expression of a DNA
sequence, said at least one vector construct comprising:
IL-12 gene in combination with B7-2 gene; IL-12 gene in combination with B7-1 gene; B7-1 gene alone ; B7-2 gene alone; or IL-12 gene in combination with B7-1 gene in combination with B7-2 gene, wherein said combination may be on one vector or on more than one vector.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the at least one vector construct comprises a polynucleotide vector.
In accordance with another aspect of the present invention, there is provided the composition wherein the at least one vector construct comprises a combination of a virus and a plasmid; or a virus and a polynucleotide; or a plasmid and a polynucleotide; or a virus and a plasmid and a polynucleotide.
5k In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the at least one vector construct comprises a virus.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the virus is fowipox.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the virus is canarypox.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the virus is adenovirus.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the virus is vaccinia virus.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the virus is swine pox virus or polio virus.
In accordance with another aspect of the present invention, there is provided the composition wherein the virus is a retrovirus.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the at least one vector construct comprises a plasmid.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the plasmid is a DNA plasmid comprising a promoter which allows for the expression of the gene or combination of genes.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the plasmid is pRc/CMV.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the hematopoietic tumour is a leukemia.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the hematopoietic tumour is a lymphoma.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the hematopoietic tuniour is a myeloma.
In accordance with another aspect of the present invention, there is provided the coniposition of the present invention wherein the leukemia is acute lymphoblastic leukemia (ALL), acute myelocytic leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL) or juvenile myelo-monocytic leukemia (JMML).
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the lymphoma is B-Cell Burkitt's lymphoma, Hodgkin's lymphomas, non-Hodgkin's lymphomas, Ki-1 positive anaplastic large cell lymphomas, T cell lymphomas or histiocytic lymphomas.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the myeloma is a multiple myeloma.
In accordance with another aspect of the present invention, there is provided the pharmaceutical composition for use as a vaccine for reducing hematological tumour burden in a mammal.
In accordance with another aspect of the present invention, there is provided the pharmaceutical composition for use as a vaccine wherein said vaccine is suitable for administration intravenously, intradermally, orally, intranasally, subcutaneously, intranodally, intralymphatically, or intrathecally.
In accordance with another aspect of the present invention, there is provided the pharmaceutical composition for use as a vaccine wherein said vaccine is suitable for administration at the site of the hematological tumour.
In accordance with another aspect of the present invention, there is provided the pharrr-aceutical composition for use as a vaccine wherein said vaccine is suitable for administration at a site different from the hematological tumour.
5m In accordance with another aspect of the present invention, there is provided the pharmaceutical composition for use as a vaccine wherein said vaccine is suitable for administration at a site distant from the hematological tumour.
In accordance with another aspect of the present invention, there is provided the pharmaceutical composition in the preparation of a medicament or a medicine wherein said medicine or medicament is suitable for administration intravenously, intradermally, orally, intranasally, subcutaneously, intranodally, intralymphatically, or intrathecally.
In accordance with another aspect of the present invention, there is provided the pharmaceutical composition in the preparation of a medicament or a medicine wherein said medicine or medicament is suitable for administration at the site of the heniatological tumour.
In accordance with another aspect of the present invention, there is provided the pharmaceutical composition in the preparation of a medicament or a medicine wherein said medicine or nledicament is suitable for administration at a site different from the hematological tumour.
In accordance with another aspect of the present invention, there is provided the pharmaceutical composition in the preparation of a medicament or a medicine wherein said medicine or medicament is suitable for administration at a site distant from the hematological tumour.
Brief Description of the Figures Figure 1. Indirect immunofluorescence profiles of A20 parental and variant lines. A20 parental and variant lines were stained for B7-1, B7-2, and MHC class I and II
molecules.
Stippled lines represent staining with isotype controls; Solid lines represent staining with the primary mAb followed by secondary FITC goat anti-rat (B7-l and B7-2) or streptavidin (MHC I and MHC II) fluorescent conjugate. The FITC goat anti-rat conjugated antibody alone was used as a control for IgG staining.
Figure 2. Cytokine production of purified allogeneic CD4+ T cells to A20 variants.
CTLL proliferation in response to IL-2 in supematants derived from allogeneic mixed lymphocyte tumour cultures (A), CT.4S proliferation in response to IL-4 in supernatants derived from allogeneic mixed lymphocyte reactions (B), IFN-y production by CD4+ T
cells to A20 variants as assessed by ELISA (C).
Figure 3. IL-2 production is inhibited by anti-B7-1 in mixed lymphocyte tumour cultures. Monclonal antibodies 1610A1 completely inhibits IL-2 production in mixed lymphocyte cultures consisting of purified allogeneic CD4+ T cells and A20/B7-1 (A) or A20/B7-1 /mL-12(B).
Figure 4. Percent Survival of animals injected with A 20 and variant cell lines. Kaplan Meier survival statistics were performed as described in Materials and Methods. Events were recorded when animals were euthanized for tumours that had reach 2 cm in diameter or that had ulcerated. Survival for A20/B7-1, A20/mIL-12, and A20/B7-1mIL-12 are superior and statistically significant (p<0.005) when compared pairwise with the A20/pRc/CMV vector control.
Figure 5. Percent Survival of animals injected with A20 variants and A20 parental cells at different sites and at the same time. Animals were inoculated with 105 variant cells on one flank and 105 variant cells on the opposite flank. Survival, was monitored. Log rank pairwise comparison demonstrated that only survival for the A201137-1/mYL-12 treatment group was statistically significant when compared to the A20/pRc/CMV or A20/pRc/pac (p<0.02). Both the A20/[n1L-12 and A201137-1/mIL-12 treatment groups had better survival than the animals inoculated with the A20B7-1 variant and the unmodified parental cells (p<0.44).
Figure 6. Percent Survival of animals inoculated with a mixture of 10' parent and 10$
variant lines at the sarne site. Log rank pairwise comparison demonstrated that both the A20/rnIL-12 and the A20/B7-1/rcdL-12 treatment groups had better survival than the A20/pRc/CM't% treatment group (p<0.0001). The survival of the A,20/H7-1/mIL-12 treatment group was also statistically superior when compared to the A20/pRc/pac treatment group (p<0_001), the A20B7-1 group (p<0.0001), and the A20/mIL-12 treatment group (p<O.OOS).
Figure 7. Generation of CTLs in animals imrnuwxized with the A20/rn.IT; 12 or ]/mIL-12 variant lines and subsequently challenged with the A20 parental cell line_ A
syngenic mixed lymphocyte tumour culture was perfurmed against the irradiated parental line by usina spleen cells as responders from mice that had previously been immunized with the A20/nmlL-12 or A20/87-1/mYL-12 variant lines and subsequently challenged with the A20 parental line. After 6 days of in vitro stimulation, lytic activity (as measured by DNA fragmentation) against the 3H-thymidine labeled parental A20 was assessed in a 4 h JAM assay.
Figure 8. Indirect Immunofluorescence profiles of A20 parental and variant lines. A20 parental and variant lines were stained for B7-1, B7-2, and MHC class I and II
molecules.
Solid lines represent staining with isotype controls; stippled lines represent staining with the primary mAb followed by secondary FITC goat anti-rat (B7-1 and B7-2) or streptavidin (MHC I and MHC II) fluorescent conjugate. In this figure, A20/B7-denotes clone A20/B7-2-3. It should be noted that the increase in 87-2 expression in the other A20/B7-2 clones used in various experiments conducted is comparable.
Figures 9. Percent Survival of animals injected with A20 and variant cell lines. 8-10 wks. old female BALB/C mice were subcutaneously inoculated with 105 cells of the parental or variant A20 cell lines on the right flank and survival was subsequently monitored. Kaplan Meier survival statistics were performed as described in Materials and Methods. Events were recorded when animals were euthanized for tumours that had reach 2 cm in diameter or that had ulcerated. Survival for A20/B7-2 clones 3 and 5, A20/mIL- 12, or combinations of A20/B7-2 clones and A20/mIL- 12 are superior and statistically significant (p<0.05) when compared pairwise with the A20 or A20/pRc/pac vector controls.
Figure 10. Percent survival of animals injected with A20/B7-1 and A20/B7-2 variant cell lines. 8-10 wk. old female BALB/c mice were subcutaneously inoculated with 105 cells of the indicated A20 cell variants and survival was monitored. Kaplan Meier survival statistics were performed as described in Materials and Methods.
Events were recorded when animals were euthanized for tumours that had reached 2 cm in diameter or that had ulcerated. Survival for A20/B7-2 clone 3 is superior and statistically significant (p<0.0005) when compared pairwise with the A20/pRc/CMV or A20/B7-1 variant cell lines.
Figure 11. Immunofluorescence staining of tumour cells derived from an animal inoculated with the A20/B7-2 variant. The tumour cells derived from mouse #3669 which had developed and aggressive tumour after inoculation withA20/B7-2-5, the A20 parental cell line, and the A20/B7-2 variant were stained for B7-2 as described in Materials and Methods.
Figures 12. Percent Survival of animals injected with A20 cell variants in a minimal disease model. The A20 parental cells were subcutaneously injected on the left flank. 24 h later the A20 variant cell lines were injected on the opposite flank..
Kaplan Meier survival statistics demonstrated that only the animals inoculated with the clone had a statistically significant increase in survival when compared pairwise with the A20/pRc/CMV (p<0.006) and A20/pRc/pac (p<0.02) vector control groups.
Figure 13. Percent Survival of A20 variant cells in BALB/c nu/nu mice. 105 A20 parental or variant cells were injected subcutaneously and survival was monitored.
Statistically significant differences were not demonstrated when each of the groups were compared pairwise p<0.05.
Figure 14. Generation of CTLs in animals inununized with the A20 variant cell lines.
BALB/c mice were inoculated subcutaneously with 105 A20 parental or variant cell lines.
After 16 days, spleens were harvested and cell suspensions prepared. These lymphocytes were cultured with irradiated A20 cells. After in vitro stimulation, lytic activity (as measured by DNA fragmentation) against the 3H-thymidine labeled parental A20 was assessed in a 4 h JAM assay.
Figure 15. Tumour regression in mice upon termination of CD8+ T cell depletion.
Groups of 5 mice each were depleted of CD8+ T cells 105 A20 tuniour cell variants were subcutaneously inoculated. By day 19, 3 of the mice inoculated with the A20/B7-variant developed progressive tumours. CD8+ T cell depletion was terminated on day 29. By day 37, only one mouse had a tumour while the tumours had regressed in the remaining mice. As previously shown, mice depleted of CD8+ T cells had inoculated with A20B7-1/mIL-12 variants did not develop tumours.
Figure 16. LAK cell lysis of A20 parental and variant lines. LAK cell cultures were prepared from naive BALB/c mice as described in Materials and Methods. 51 Cr labeled cell lines were used as targets. % NK cells as measured by the DX5 market was 42.6%.
CD3 contamination was 0.4%. LAK cell mediated lysis of A20/B7==1 and A20/B7-2 are compared in (A). LAK cell mediated lysis of A20/B7-1 /mIL-12, A20/mIL-12 and A20/B7-2 are compared in (B).
Figure 17. Lysis of A20 targets by NK cells isolated from mice i.v. immunized with the A20 transfectant cell lines. BALB/c nulnu mice were inoculated with A20/pRe/pac, A20/B7-2, A20/B7-1/mIL-12, A20/mIL-12 variant cell lines. Spleens were removed, single cell suspensions were prepared, and NK cells were enriched as described in Materials and Methods. CD3 contamination was < 1% and DX5 staining ranged from 69-77 %. Labeled A20 target cells were than co-cultured with isolated NK cells at varying E:T ratios.
Detatded Description of the Preferred Embodiments Definitions The term "adenovirus" as used herein mean.s any of a group of viruses having the characteristics of an adenovirus. Adenoviruses aze genErally characterized as double-stranded DNA viruses. They have icosahedral capsids with twelve vertices an,d seven surface proteins. The virion is non-enveloped, spherical and about seventy to ninety nm in size. The genome of an adenovirus typically encodes about thirty proteins.
Both strands of adenovzrus DNA encode genes. Transcriptxoxx occurs in three stages -immediate early, early and late.
The tenn "canary pox virus" as used herein means a poxvirus of the genus avipoviras.
Pox viruses can infect many species of birds, and each species of bird may have its own unique species of pox virus (nnynah bird pox virus, canary pox virus, etc_).
The term "fowl pox" as used herein means a poxvirus infection of poultry and other birds characterized by the formation of wart-like nodules on the skixi and diphtheritic necrotic masses (cankers) in the upper digestive and respiratory tracts. The term "fowl pox virus" as used herein means the type species of the genus avipovirus. It is tlie aetiologic agent of fowl, pox.
The term " hematological" as used herein means of or relating to or involved in hematology. "Hematology" as used herein means the brancb of medicine that deals with diseases of the blood and blood forming orgaits.
lIa The term "hemato,poietic" as used herein means pertaining to the formation of blood or blood cells_ The terxza, "intradermally" as used herein means administered into or within the skin.
The term "intranasally" as used herein rneans administered into or within the nose.
The term "intrathecally" as used herein. means administered into or within the fluid-filled spaces between the thin layers of tissue that over the brain and spinal coi-d.
The term "leukaemia" as used herein means a malignant neoplasm of blood-forming tissues charactexized by abnormal proliferation of leukocytes. "Acute leukaemia" as used herein means rapidly progressing leukemia. "Chronic leukaenua" as used herein means slowly progressing leukemia. "Myelocytic leukaemia" as used herein means a malignant neoplasm. of blood-forming tissues marked by a proliferation of xnyelocytes and their presence in the blood. "Lymphocytic leukaemia" as used herein means leukaemia characterized by enlargement of the lyr,rxphoid tissues and Iymphocytic cells in the circulating blood. "Myeloid leukaernia" as used herein means a chronic leukaemia characterized by granular leukocytes. "Lymphoblastic leukaemia" as used herein mean.s a form of lymphocytic leukaemia in which the abnormal cells in the circulating blood are almost totally lymphoblasts (immature white blood cells). "Juvenile myel.o-monocyti.c leukae.rnia" as used herein means a very rare type of childhood leukemia. It is also sometimes called chronic myclogenous leukaemia.
The term "lymphoma" as used herein means a neoplasm of lymph tissue that is usually malignant. There are many types of lymphoma: "Hodgkin's lymphoma" and Nan-hlodgkins lymphomas being two types. The primary difference between Hodgkin's 1lb Lymphoma and Non-Hodgkin's Lymphoma is the presence of a specific abnormal cell, namely, Reed-Stera;berg cells. There are diff.erent names for Hodgkin's disease:
lymphocytic predominance, nodular sel.erosis, mixed cellularity, ly-mphocyte depletion and unclassifxed.
Non-Hodgkin's Lymphoma (NHL) occurs with the malignant (cancerous) growth of B or T cells. Although both types can develop into lymphomas, B-cell lymphomas are much more common than T-cell lymphomas. There are at least 29 different types of NHL each differentiated by the type of cancer cell. Some scientists classify cells by gi:owth rate: indolent refers to slow growth and aggressive refers to fast-growinb cells.
Some also classify NHLs by cell type: T-cel1, B-cell and follicular cell, etc_ "Burkitt's Lymphoma" as used herein means a type of non-Hodgkin.'s lymphoma that most aften occurs in young people between the ages of 12 and 30.
"Anaplastic large cell lymphoma" as used herein means a rare aggressive form of lymphoma that is usually of T cell origin. "Histiocytic lymphoma" as used herein means a malignant tumor of ret-icular tissue.
The term "myeloma" as used herein means a tumor of one or more cells of the bone marrow. Mycloma, is also known as "multiple myelorna" and myelomatosis.
It is a type of cancer arising from plasma cells, which are normally found in the bone marrow.
In rayeloma, one plasma cell becomes defective and multiplies rapidly, to produce too many plasma cells.
The term "orally" as used lrereiuo means administered into or within the rnouth.
The term "polio virus" as used herein means the virus causing poliornyelitis.
"Poliomyelitis" as used herein means an acute viral disease marked by inflammation of nerve cells o the brain stem and spinal cord.
The term "swine pox" as used herein means a variety of the chicken pox, with acuminated vesicles containing a watery fluid. Also referred to as the water pox. Swine pox vinis is a genus of the family Poxv;rirlae, subfamily Chordopoxvirinae, containuing one species, swinepox virus.
The term "tumor burden" as used herein means, the number of cancer cells, the size of a tumor, or the amount of cancer in the body of a mammal or human, "Upregulation" means gene expression levels are increased.
The term "vaccinia rrirus" as used herein means a virus strain used to vaccinate against smaUpox.
Materials and Methods for IL-12 and/or B7-1 Mice. Female BALB/c, C57BL/6, and nu/nu mice 8-10 wk. of age were purchased from Charles River (St. Constant, Que.) and housed at the Animal Facility at the University of Toronto Medical Sciences Building. All experiments were conducted in accordance with the University of Toronto Animal Care guidelines.
Tumour Cells. A20 is a BALB/c B cell lymphoma (Kim et aL, 1979) expressing MHC class 1 and class iI H-2d molecules and was purchased from the ATCC.
Tumour cells were cultured in vitro in RPMI 1640 medium, supplemented with 15% FCS, 5X10,5 lld M 2-ME. Cells were periodically tested for and found to be free of mycoplasma contamination.
DNA-medialed gene transfer. The pRc/CM'V-B7-1 plasxnid was kindly provided by Dr. L. Nadler (Dana-Farber Cancer Institute, Harvard Medical Sehool, MA) and contains the murine 137-1 cDNA in the pRc/CMV expression vector which includes the bacterial neomycin phosphotransferase (neo) gene eonferring resistance to G41$_ A
BioRad'rA
electroporator was used to tran.sfect 20 ug of linearized pRc/CMV-B7-1 cDNA
(digested with Pvu 1) into the A20 B cell lyrnphoma,. The vector coritrol, pRc/CMV, was also linearized with Pvu I and transfected into A20 cells. Selection in G418 at a final coneentration of 500 uglml was initiated 48 h after tcansfeetion. Positive transfectants were further cloned by limiting dilution.
Recombinant IL-12 retrovirus. To achieve simultaneous expression of the 40-kDA
(p40) and. 35-kDa (p35) subunits of mIL-12 as well as a dominant selectable marker from a single retroviral vector, an expression cassette consisting of the mIL-12 p40 and p35 cDNAs (provided by Genetics Institute, Cambridge, MA) (Hawley et al., 1994), separated by a picornavirus inte,rr.xal ribosome entry site (provided by J.
Majors, Washington University, St. Louis, MO) (Ghattas et al., 1991; Hawley et al., 1996) was inserted into the MSCVpac retroviral vector containing the puromycin 1V=acetyl-transferase (pac) gene (Hawley et al., 1994). In the resulting construct, termed MSCVpac-mIL-12, the p40 and p35 cDNAs are under the transcriptional control of the viral long terminal repeat while the pac gene is transcribed from an internal phosphoglycerate kinase promoter. Construction details and the characterization of the MSCVpac-mlL-12 retroviral vector will be reported elsewhere (Lieu et al., 1997)_ Linearized MSCVpac-mlL-12 plasrnid DNA. was electroporated into GP+E-8 6 ecotropic helper-free packaging cells (Markowitz et al., 1988). Viral supernatant was collected 24 h later and used to transduce tunacamycin-treated (0.1 ug/ml for 16 h) GP+E-86 cells (Hawley et al., 1992) and stable transductants were obtained by selection in 1 ug/mL puromycin (ICN Biomedicals, Costa Mesa; CA) for 2 wk. The pooled population of stably-transduced GP+E-86 cells (GP+E-86/MSCVpac-mlL-12) produced recombinant IvISCVpac-mIL-12 at a titer of 8 X 105 puromycin-resistaut C1;U/ni.l when assayed on NIH3T3 fiibroblasts. The corresponding NIH3T3 titer of parentallvlSCVpac virus exported froin a pooled population of stable GP+1r-86 transductants was puromycin-resistant CI'Ulm1. Producer cells were propagated in DME witlt 4.5 g/1 glucose supplemented with 10% calf serum (Life Technoloaies, Gaithersburg, MD) in a humidified atmosphere containing 5% C02 at 370 C.
Viral supernatants for transduction of A20 tumour cells and B7-1 transfectants, were collected from confluent GP+E-86 cultures 24 b after the medium was changed to RPMI
1640 supplemented with 50 uM 2-ME and 15% FCS, centrifuaed at 3000 g for 5 minutes to remove cellular debris, filtered through 0.45 um filters, and used immediately or frozen at -700 C for later use. To generate stable tumour cell transductants constitutively expressing exogenous mIL-12, tun.tour cells growing at a density of 2 X 105 cells/ml were incubated with 6 nzL viral supernatant in the presence of 2 ug/ml polybrene (Sigma, St. Louis, MO). Twenty-four hours later, an additional 6 rnl viral supexnatant containing 2 ug/mi polybrene was added and the incubation continued for a further 24 h.
The znedium was then charnged to fresh RPMI 1640 supplemented with 50 uM 2-ME and 15%
FCS, and 3 ugfrrtl puromycin was added 48 h later.
,lmmunostai-ntng and FACS analysis. Trans.fectants were analyzed with mAb 1.Cx10 (Nabavi et al., 1992) which is a rat IgG2a B7-1- specific mAb purcliased frorn PharMingen (San Diego, CA). A20 parental and variant lines were also stained for GL1 (Freeman et al._, 1993) a rat IgCr2a 87-2 specific mAb, MK-D6 (Watts et al., 1984) an azxti I-Ad r-mAb, 34-1-2S (Ozato et al., 1982) an anti. H-2 TCdDd mAb. These antibodies were purified from culture supematants using protein Cr sepharose=m purchased from Pharmacia (I'iscataway, NJ). After purification, both MK-D6 and 34-1-2S were biotinylated. Cell lines were also stained with FITC-conjugated anti-mouse IgG
(g-chain specific) purchased from Sigma ImmunoChemicals (St Louis, MO). Isotype controls included biotinylated L243 (Lampson et al., 1980) a mouse anti-human Ia, IaG2a antibody, and rat IgG2a isotypic control from CedarLane Laboratories (Hornby, Ontario).
FITC conjugated goat anti-rat IgO purchased froin CedarLane Laboratories (Flornby,Can.) was used for detecting the binding of B7-1 and B7-2.
Bioti.nyated antibodies were detected using FITC conjugated streptavidin purchased frorrt Molecular Probes (Eugene,OR). Stained cells were analyzed on a Becton DickinsonTM flow cytometer.
Mixed Lymphocyte Tumour Czslt rtres. MLR cultures between C57BL/6 lymph node T
cells and the A20 lymphoma variants were prepared as follows. Lymph nodes taken frorn the ingui.nal, axillary and salivary sites were used to raake suspensions of responder cxlls in HBSS supplemented with 5% FCS, and penicillin/streptomycin. Following their elu.tion from a SephadexTM G-10 column, the cells were treated with a cocktail of 1VIAbs wbich included: 3.168 (anti-CD8), 53.6.7 (agti-CD8), RA3-6B2 (anti-B220), (anti-HSA), and Y-3P (anti-A6) all purchased from ATCC. With the exception of 3.168 a11 supernatants obtained from hybridomas rurchased froin the ATCC were purified using protein A or O SepharoseTM ;purchased from Pharmacia (Piscataway, NJ).
After Ab and complement treatment (Low-Tox-MTM, CedarLane, Hornby, Ontario) the viable cells were recovered by buoyant density centrifugation using Lyxnpholyte-Mm obtained from CedarLane ()`Iornby, Ontario). T cell purity as demonstrated by immunofluorescence staining ranged from 90%-95%. In addition, no response was obtained with T
cells plus Con A alone, indicating that APC contamination was minimal (data not shown).
Stimulator A20 variants were irradiated with 10,200 rads and washed once before use.
IV1LR cultur.es of 1X105 T cells and 2X105 A20 variant cells in a total volume of 200 ul RPMI 1640 mediurn supplemented with 10% FCS purchased frorr.x P.A. Biologicals (Sydney, Australia), non-essential amino acids, glutamine, pyruvate, penicill,inlstreptorrtycin, and 2-ME, were prepared in duplicate using 96 well plates (Nunc, cat. 1-67008). The cultures were incubated for 3 d at 370C and 5% C02 in humidified conditions, at which time 50 ul of inediLun was removed and replaced with 50 ul of fresh medium. 48 h later, the contents of duplicate cultures were pooled, centrifuged, and 300 ul of supernatant was collected and frozen at -70oC.
Bioassays. Bioassays for both IL-2 and IL-4 were performed identically with the exception that IL-2 was detected using the CTLL cell line and IL-4 was detected using the CT.4S cell line (Hu-Li, et al., 1989). The CT.4S cell line was kindly provided by Dr.
W. Paul (National Institutes of Health, Bethesda, MD). Anti-IL-4 mAb 11B11 purchased from the ATCC, used at 4 ug/ml was added to all cultures containing CTLL
cells. Both the CTLL and CT.4S lines were maintained in continuous culture using rIL-2 supernatant or rIL-4 supernatant, respectively, derived from the lymphokine-secreting X63 cell line series, as developed by Kanagawa and Melchers (Kanagawa and Melchers, 1988).
Two-fold dilutions of supematant, starting at 1/4 were prepared in triplicate and indicator cells were cultured for 24 h in 100 ul using 96 well plates. During the last 8 h, the cells were labeled with 3H-thymidine (Amersham, 5 Ci/mmol), added at I uCi per well. Cultures were processed for liquid scintillation counting and the amount of incorporation was determined using the Packard Top Count system. For inhibition of cytokine production, anti-B7-1 mAb 1610A1 kindly provided by Hans Reiser (Dana-Farber Cancer Institute, Harvard Medical School, MA), anti-B7-2 GL 1(ATCC), or control rat Ig purchased from Sigma (St.Louis, MO.) or control hamster Ig purchased from Jackson (West Grove, PA) were added to the cultures.
ELISAs. Mouse IL-12 was quantified by using the ELISA mouse IL-12 Cytoscreen kit (Biosource, Camarillo, California) and performed as described by the manufacturer's instructions. 1 X 106 of variant cells were plated in complete media and supernatants that were used for the ELISA were collected 24 h later. Supernatant levels of IFN-y were measured by capture ELISA using anti-murine IFN-y MAbs purchased from PharMingen (San Diego, CA). ELISA was performed as described by the manufacturer's instructions, using the diluted supematant.
Animal Studies. For tumourigenicity experiments, A20 parental and variant lines in log phase were harvested, washed, and resuspended in 0.2m1 RPMI 1640 medium lacking any supplements. 105 cells were injected subcutaneously in the right flank.
For challenge experiments, animals found free of tumour were either boosted and then challenged 10 days after the boost or challenged without boosting with 105 A20-parental cells injected subcutaneously on the left flank. In minimal disease inodel mixing experiments, 105 A20 parental and 105 A20 variant cells were mixed and injected subcutaneously at the same site (right flank) or at different sites (right and left flank). In all animal studies, 10 ear-tagged animals were injected for each variant cell line. 5 animals each were injected to assess the tumourigenicity of the A20/pRc/pac and A20/B7-1/pac vector controls. Tumour incidence and size were monitored 3 times a week. Animals were euthanized when the tumour diameter was approximately 2 cm or tumour ulceration was apparent.
For injections into nu/nu mice, 105 A20/pRc/pac vector control, A20/mIL-12, and A20/B7-1/mIL-12 variants were subcutaneously inoculated. Five animals were used for each variant group.
Jam Test. The Jam CTL test was performed as described by Matzinger (Matzinger, 1991). Briefly, single cell suspensions were made from spleens derived from mice that had been previously immunized with the IL- 12 and B7-1 /IL-12 variants and had furthermore rejected challenge by the parental A20 unmodified line. These responder cells were incubated with irradiated A20 parental cells for 6 days. Viable effector cells were then incubated with 3H-thymidine (Amersham) labeled A20 target cells at different E:T ratios in a 96 well plate (Nunc, cat. 1-63320) for 4 h at 37 oC. After 4 h, the cells were harvested with a cell harvester and counted by scintillation counting. %
specific killing was calculated as follows: (Spontaneous cpm-Experimental cpm)/(Spontaneous cpm) X 100. Cpm were taken of target cells alone both at to and t4 to assure that target cells were not undergoing spontaneous cell death. The standard error of the mean did not exceed 5.5 %. As controls, C57BL/6 mouse spleens were used as responders and BALB/c mouse spleens were used as stimulators in an allogeneic mixed lymphocyte reaction. Naive BALB/c mouse spleens incubated with the parental A20 cell line was used as a negative control. In addition, animals were immunized with the A20/pRc/pac vector control and two weeks later the spleens were dissected and used for JAM
assays.
Animals immunized with the A20/pRc/pac vector control at the same time as the variants could not be assessed because these vector control animals had to be euthanized within three weeks of the experiment reaching tumour diameters of 2 cm.
In-vivo Depletion studies. Ascites fluid of hybridoma 2.43 (anti-CD8) (ATCC;
TIB
210), and GK1.5 hybridoma, L3T4) (ATCC; TIB 207) were kindly provided by Dr.
K.
Irvine (National Cancer Institute, Bethesda, MD). BALB/c mice were i.v.
injected with empirically detennined levels of 2.43 and GK1.5 diluted in 0.5 mL of PBS four days prior to tumour inoculation and then weekly i.p. thereafter (Rao et al., 1996). Inguinal, axillary and salivary lymph nodes were dissected from control animals to assure >90%
depletion of T cell subsets using indirect immunofluorescence staining with FITC
conjugated mAb CD4, CD8 antibodies purchased from Pharmingen (San Diego, CA), or goat anti-rat FITC purchased from CedarLane (Hornby, Can.). The tumour cell variants were inoculated four days after the initial antibody injection.
Statistical Analysis. Kaplan Meier survival curves were compared using the log rank test in the SPSS 6.1 Macintosh Version statistical package. Differences are considered statistically significant when p<0.05.
Results for IL-12 and/or B7-1 Generation of A20 variants. The A20 murine B-cell lymphoma is IgM-, IgG+, IgA-, Ia+, Fc receptor+, and complement receptor- (Kim et al., 1979). A20 also expresses both MHC class I and class II molecules, and low levels of B7-2 as determined by indirect immunofluorescence staining (Fig.l). B7-1 expression, however, is undetectable. A20 variants expressing both low and high levels of B7-1 were generated by transfection with the murine B7-1 cDNA and selection in G418. Vector control variants were generated by transfection with pRc/CMV. Positive independent clones were obtained by limiting dilution. Because of the reported in vitro enhancement of B7-1 costimulation of T cells by IL-12 (Gajewski et al., 1995), we also introduced mIL-12 using the MSCVpac retroviral vector system (Hawley et a1.,1994). Preliminary studies had shown no significant anti-tumour effects using the A20/B7-1-low expressing variant, hence, the populations used secreted mIL-12 at levels of 1, 250 pg 7.81 pg /ml/106 cells/24 h.
Immunostaining and FACS analyses demonstrated that expression of the costimulatory molecules, B7-1 and B7-2, as well as MHC class I and class II, were stable in all variant lines (Fig. 1). The phenotypic profiles of these variants did not charige upon reassessment after several weeks in culture. In addition, the doubling rates of the A20-parental and variant lines were not significantly different (data not shown).
Costimulation and Cytokine Production ofAllogeneic CD4+ TLymphocytes by A20/B7-1 and A20/B7-1/mIL-12. T'o assess the costimulatory function of the A20 variants, A20 parental and variant lines (H-2d) were compared in their ability to activate purified allogeneic CD4+ T cells in a mixed lymphocyte tumour culture assay, and IL-2, IL-4, and IFN-y secretion by the reacting T cells was assessed. Only the (high) and A20B7-1 /mIL-12 variants were able to stimulate allogeneic (H-2b) CD4+ T
cells (Fig. 2 A, B) to secrete IL-2 and IL-4. IL-2 or IL-4 secretion by allogeneic T cells was not seen by the A20/B7-1 (low), A20/mIL-12, or parental tumour lines despite the low endogenous level of B7-2. Interestingly, both the levels of IL-2 and IL-4 secretion by T cells was reduced in the A20/B7-1 /mIL-12 mixed lymphocyte tumour culture as compared to the culture containing the A20/B7-1 variant.
IFN-y secretion was also assessed after stimulation of allogeneic CD4+ T cells with the A20 parental and variant lines. Only A20/mIL-12 or A20B7-11mIL-12 variants were able to induce allogeneic T cells to secrete IFN-y (Fig. 2 C). Both variants induced the T
cells to secrete comparable levels of IFN-y.
IL-2 secretion was completely inhibited when anti-B7-1 was added to the mixed lymphocyte tumour cultures consisting of purified allogeneic CD4+ T cells and 1 or A20/B7-1/mIL-12 variant lines (Fig. 3). Anti-B7-2 had no or minimal effects on reducing IL-2 secretion when added to either the A20/B7-1 or the A20/B7-1/mIL-cultures respectively. Similar elimination of IL-4 secretion was seen when antibodies to 137-1 were added to the mixed lymphocyte tumour cultures (data not shown).
A20 Transfectants Expressing B 7-1 Delay Tumour Onset and Prolong Survival While A20/mIL-12 or A20/B7-1/mIL-12 are Rejected by BALB/c Mice. 105 cells of each variant line were injected subcutaneously into the right flank of BALB/c mice. While animals receiving A20B7-1 cells develop tumours, the tumour onset is delayed. That is, 80% of animals receiving control A20/pRc/CMV cells have palpable tumours by day 18 post injection, while only 40% of animals receiving the A20/B7-1 transfectants have palpable tumours at this time point (data not shown). In addition, B7-1 prolongs survival (Fig.4).
While 50% of the animals receiving the A20/pRc/CMV vector control variant were euthanized by day 27, 50% of the A20/B7-1 recipients were euthanized by day 37.
Strikingly, the animals receiving the A20/mIL-12 or A20/B7-1 /mIL-12 variants do not develop tumours (Fig. 4). These results suggest that although B7-1 reduces the tumourigenicity of the A20 line, expression of IL- 12 or co-expression of B7-1 results in complete rejection.
Inoculation of 1'umour cells Expressing mIL-12 With or Without B7-1 Protects Against Subsequent Challenge With Unmodifted Parental Tumour Cells. Animals from the above experiment that received 105 A20/B7-1 /mIL- 12 or A20/mIL- 12 did not develop tumours. 45 days post initial injection, 50% of the tumour free animals were boosted with either the A20/87-1/mIL-12 or the A20/m1L12 variant respectively at the same site of initial subcutaneous injection. Ten days following the boost, all tumour free animals boosted and non-boosted were challenged subcutaneously on the alternate left ilank with 105 parental A20 nontransfected cell line. Tumour-challenged mice were observed for tumour incidence and survival. At this dose, both A20./B7-1/mIL12 and A20/mIL- 12 were equivalently able to protect mice against subsequent tumour challenge.
By day 41, post tumour challenge, one mouse boosted with the A20/mIL- 12 variant developed a tumour, while two mice boosted with the A20/B7-1/mIL-12 variant developed tumours. Also, one non-boosted mouse from the A20/B7-1/mIL-12 group developed a tumour. It should be noted that these tumours developed on the site of challenge with the parental cell line, i.e., the left flank. Overall, 70% and 90% of the immunized mice from the A20/B7-1 /mIL-12 and A20/mIL- 12 groups respectively, remained tumour free after having been re-challenged with a lethal dose (105 cells) of the unmodified parental line (data not shown).
A20/B7-1/mIL-12 Variants Result in Prolonged Survival In a Minimal Disease Model. In this series of experiments analogous to the clinical setting of minimal disease, mice were immunized with genetically modified tumour variants on the same day as the parental tumour cells were inoculated. Immunizations were performed at the same site (intratumourally) or at distant sites. Figure 5 shows the results of one such experiment where 105 A20 parental cells were injected into the left flank, and 105 cells of a particular A20 variant line were injected into the right flank. Figure 5 illustrates that by day 46 only one animal remained in the pRc/CMV vector control group, while no animals receiving the pRc/pac cells were alive. Also, protection was not observed in animals that were immunized with A20/B7-1 on the opposite flank. Tumours developed in both flanks in all of these animals. In contrast, both the A20/mIL- 12 and I/mIL-12 variants delayed tumour growth of parental A20 cells at the remote site.
However, only the A20/87-1 /mIL- 12 vaccination induced statistically significant increases in survival when compared to the vector control treatment groups (p<.02).
Resultant tumours in these treatment groups grew only in the flank receiving the A20 parental cells.
The above experiment was modified by inoculating a mixture of the variant tumour cells used for immunization with the parental tumour cells so as to resemble the clinical situation of intratumoural vaccination into a site of minimal disease. 105 A20-parental and 105 A20 variant lines which included A20/pRc/CMV, A20/pRc/pac, A20/B7-1, A20/B7-1/pac, A20/87-1/mIL-12, and A20/mIL-12 were mixed and injected subcutaneously into the right flank and tumour incidence and survival were monitored.
Progressive tumours grew in all animals receiving vector transduced control or the B7-1 cells and A20 parental cells at the same site. Under these conditions, the variant did not produce a delay in tumour onset (data not shown). Similarly, by day 25, two out of ten animals receiving the A20/pRc/mIL- 12 variant had palpable tumours and by day 42, 80% of these animals had progressive tumours. At the day 42 time point, 100% of the animals inoculated with the A20/B7-1/mIL-12 variant were tumour free. By day 66, only 2 tumour free mice remained in the A20/mIL- 12 treatr.nent group while 80%
of the animals receiving the A20/B7-1 /mIL-12 variant were alive and 6 out of these 8 animals were tumour free (Fig. 6). These results suggest that at these doses, the A20/B7-I/mIL-12 variant was most effective in preventing outgrowth of unmodified parental tumour cells when introduced into the vicinity of the parental tumour cells at the same site of injection.
Mechanisms Mediating Anti-tumour Immunity. Animals that had been initially immunized with 105 A20/B7-1/mIL-12 or 105 A20/mIL-12 variant lines subsequently rejected challenge with 105 parental unmodified A20 cells on the opposite site. To gain insight into the mechanism responsible for this observed in vivo anti-tumour activity, cytolytic T cell activity was assessed in these animals and compared to that in naive animals or animals immunized with the A20/pRc/pac vector control. Results demonstrated that animals immunized with the A20/mIL-12 variant had a specific lysis of A20 targets of 76.8%, while animals immunized with the A20/B7- 1/mIL- 12 variant had a lysis of 72.8% both at an E:T of 40:1 (Fig. 7). At this ratio, animals immunized with the A20/pRc/pac variant or naive mice demonstrated less than 50% of this lytic activity against the A20 parental line.
To gain insight into the particular T' cell subsets involved in mediating anti-tumour immunity, BALB/c mice were depleted of CD4+ or CD8+ T cells and inoculated with the A20/pRc/pac, A20/mIL-12, and A20/B7-1/mIL-12 variant tumour lines. By day 27, tumours had developed in untreated animals or animals depleted of CD4+ or CD8+
T
cells inoculated with A20/pRc/pac tumour cells (Table 1). Only 2/5 animals depleted of CD4+ T cells and inoculated with the vector control, A20/pRc/pac, developed tumours.
It should be noted, however, that statistically significant differences in survival, are not achieved in the untreated or depleted animals that have been inoculated with the vector control. All animals receiving the IL-12 or B7-1 and IL-12 variants, however, did not develop tumours suggesting that the enhanced immunogenicity induced by the IL-variants could be mediated by both CD4+ or CD8+ T cells or by cells other than T
lymphocytes. Another tumourigenicity experiment was performed using mice deficient in both CD4 and CD8 T cells, nu/nu mice. 105 A20/pRcpac vector control, A20/B7-1, A20/mIL-12, A20/B7-1/mIL-12 variant lines were injected subcutaneously into these mice and tumour development was monitored. In this experiment enhanced immunogenicity was not seen in mice inoculated with the IL- 12 variants (data not shown). Taken together, these data suggest that either CD4+ or CD8+ T cells may mediate the enhanced immunogenicity elicited by the IL-12 variants and that this activity is lost in mice deficient in both CD4+ and CD8+ T cells.
I)iscussion for IL-12 and/or B7-1 Approaches to enhance the immunogenicity and reduce the tumourigenicity of tumours are actively being tested in various murine tumour models. These approaches have included gene transfer of costimulatory molecules such as B7-1 or various cytokines ('Tepper et al., 1989; Fearon et al., 1990; Dranoff et al., 1993; Pardoll, 1995). Several cytokines have been able to reduce the tumourigenicity of the tumour cell and even confer protection against unrnodified parental tumour cells (for review see Colombo et al., 1994). Similarly, reduction in turxmourigenicity attd protection against uni-oodified parental tumour cells has been observed through the introduction of B7-1 (Chen et al., 1992; Baskar et al., 1995; Townsend et al., 1993)_ A strategy to enhance the imxnunobenicity of the A20 murine B cell lympholna has been studied by introducing both the genes for B7-1 and IL-12 into the same tumour cells. The A20 B ce111ytnphoma murine tumour model is a good model to study such a strategy in, that it expresses IgG, N,QIC I and ir molccules ajid levels of ezzdogenous 137-2, yet, it is tumourigenic. In fact, subcutaneous inoculation of 105 tumour cells proliferate and kill rnice within 25 days.
Purified, allogeneic CD4+ T cells secreted IL-2 and IL-4 in the presence of A20 cells altered to express high levels of B7-1. The secretion of these cytokines in the presence of B7-1 is consistent with work from previous groups (Galvin et al_,1992). The specifieity of these increases was shown by elimination of this cytokine secretion when antibodies to B7-1 were added to the cultures. Interestingly, despite the low levcls of B7-2 expression by the parental A20 cells, this level of expression was not adequate to costimulate T cells to secrete IL-2 or II,-4. In addition, antibodies to B7-2 did not significantly reduce the IL-2 or XL.-4 secretion induced by the A20/B7-1 or A20B7-1/m1L-12 variants.
Because B7-2 has been shown to costimulate T cells (Chen, C. et aI.,1994), a threshold level of 87-2 expression may be necessary for this response and that A20 cells express sub-threshold levels of this molecule.
IL-12 variants either with or without B7-1 induced marked increases in XpN-y secretion by allogeneic T cells. The levels of IF'N-y induced were comparable and not statistically significantly different between the A20/mIL-12 or the A20/B7-1/mIL-12 variants indicating that the different in vivo effects between the A20/rnlL-12 or A20/B7-I/mIL-12 variants is not likely due to differences in the in vxvo levels of IL-12 secretion between these independently derived IL- 12 lines. The amount of both IL-2 and secreted by allogeneic T cells was reduced in the cultures consisting of the ,,4,20/B7-1/mIL-12 variarnts. Others have shown that IL-12 plays a role in cell-mediated immunity via the regulation of Thl and Th2 subsets (Romagnani et al., 1992; Germann et al., 1993;
Manetti et al., 1993). Moreover, IL-12 promotes the production of IFN-y both in vitro (Chan et al., 1991) and in vivo (Brunda et al., 1993). Because IL-12 has been shown to promote the generation of Th1 cells (Cardinb et al., 1989), it was not unexpected to see reduced IL-4 secretion in the presence of IL-12. The explanation for the reduction in IL-2 secretion, however, is unclear.
Indeed, the tumourigenicity of the A20 cells was reduced and mice immunized with the A20/B7-1 variant had delayed ttunour onset. These results are consistent with results expected for modification of relatively non-immunogenic tumour cells with the costimulatory molecule (Chen, L. et al., 1994). My preliminary studies have shown that animals irnraunized with variants that express only low levels of I37-1 develop progressive tumours at a similar rate to the parental line whereas the high expressing cell line significantly delays tumour onset (data nat shown).
By direct comparison, tturnourigenicity was strikingly reduced when IL-12 was introduced into the A20 vector control and A20/B7-1 high expressing variants.
Syngeneic animals completely rejected the A20/rnIL-12 or A20/B7-1/mIL-12 modified tumour cells. 80% of these animals were protected against subsequent challenge with the parental tumour line. In addition, these data demonstrate that activation of CTLs may play a role in the marked reduction in the tumourigenicity of the A20/B7-1/mIL-12 or A20/mIL- 12 variants.
In a more stringent experiment analogous to the minimal disease state, the A20 parental cells and the A20 variant lines were injected at the same time and at the same site. Progressive tumours grew in all animals, except in those animals receiving the A20B7-1 /mIL- 12 variant.
In addition, while all other treatment groups, including the A20/B7-1 group developed tumours by day 16 post injection, tumour onset was delayed in the animals receiving the A20/mIL- 12 variant at the same site. However, most animals receiving the A20/mIL-12 tumour cells eventually developed progressive tumours and by day 66 post injection only 20% survived. At this same time point, 8 out of 10 animals inoculated with the A20/B7-1/mIL-12 variant and the parental cells at the same site were alive, and 6 of these animals were still tumour free.
In the most stringent experiment, the A20 parental, unmodified tumour cells were inoculated at the same time but at a different site than the A20 variant lines. Survival of the group that received the A20/B7-1/mIL-12 variant was superior, although not statistically greater than the animals receiving the A20/mIL-12 variant. The therapeutic advantage of these variants may be greatest in a minimal residual disease setting when variant tumour cell immunogens are inoculated into sites of residual disease.
The mechanism of the enhanced anti-tumour activity was complex and probably multifactorial. Increased CTL activity was detected in mice that survived tumour challenge with the IL- 12 or B7-1 and IL- 12 variants compared to un-immunized mice or mice immunized with vector control cells. Tumourigenicity experiments performed in nu/nu mice showed that the enhanced anti-tumour activity was lost in these mice. On the other hand, the T cell depletion experiments showed that the enhanced anti-tumour activity seen in IL- 12 or B7-1/IL-12 variants was not lost when either CD4+
or CD8+
lymphocytes were depleted from BALB/c mice. This suggested that either T cell subset could mediate this anti-tumour activity or that some non T cell such as NK
cells were participating. Recently, studies suggest that B7-1 may enhance the anti-tumour activity of NK cells (Wu et al., 1995; Chambers et al., 1996) and IL-12 is well known to increase NK activity (Kobayashi et al., 1989; Robertson et al., 1992; Naume et al, 1992; Gately et al., 1991, 1992). The in vitro experiments also showed that there was a significant increase in the cytokines IL-2, IL-4 and IFN-y secretion from CD4+ T cells responding to the IL-12 or B7-1 /IL-12 variants. Taken together these results suggest that the enhanced anti-tumour activity generated by immunization with tumour cells modified with with or without B7-1 is due to the multiple and pleiotrophic effects induced by these genetically modified tumour cells upon the immune system.
Potent anti-tumour effects of recombinant IL- 12 or localized paracrine IL- 12 expression have been reported previously (Brunda et al., 1993; Tahara et al., 1995;
Tannenbaum et al, 1996; Nastala et al., 1994; Zitvogel et al., 1995). However, the use of tumour cells genetically modified to express both B7-1 and IL-12 at low levels would appear to offer distinct advantages over the former strategy, i.e., the potential for aximizing tumour immunogenicity (Nanda et al., 1995) while minimizing any possible side-effects associated with high circulating cytokine levels (Cohen, 1995).
These data demonstrate that tumour cells modiFied with IL-12 were superior to tumour cells modified with B7-1 alone in conferring protection. Both B7-1 and IL-12 may allow for the recruitment and activation of both Thl. and Th2 lyn-lphocyte populations and subsequent alterations in the local cy-tokine profile causing the generation of cytolytic T
lymphocyte activity. In addition, these studies provide the first formal description of anti-tumour activity with tumour cells modified to express both B7-1 and secrete IL-12.
Manipulation of the cooperative mechanisms which may be triggered by the cornbination of costimulatory and cytokine activity may be a novel and effective therapeutic approach in patients with cancer. The methodology of same site versus different site illustrates the potential of these genetically modified tumour cells as anti-tumour vaccines.
Materlals and Methods for B7-2 Mice_ Same as for IL12 and/or B7-1.
Tumour Cell s_ Same as for IL 12 and/or B7-1.
Gene Transfer. The pRc/C1VIV-B7-1 plasmid was kindly provided by Dr. L. Nadler (Dana Farber Cancer Institute, Harvard Medical School, MA) and A20/B7-1, A20/pRc/CMV variants were generated by electroporation as previously described (Pizzoferrato et al., 1997). The mouse B7-2 cDNA was cloned from cellular RNA
extracted frorn, db-cAMP stimulated M12 cells (Kim et aI., 1979) by RT-PCR
using prinaers derived from the published sequence (Freeman et al., 1993): 5'-ctgtagaegtgttecagaacttaeggaa-3' and 5'- caatttggggttcaagttectteagg-3' and internal primer 5'-taaattagtta.tca.gaaa-3'. The arnplified product was subcloned into the pRc/CMV
expression vector (Xba I and Not I sites ) and sequenced to rule out PCR
errors. To ensure for appropriate protein expression, COS cells were transiently transfected using lipofectanrzineT~4 (GibcoBRL, Life Technologies, Gaithersbuarg, MD) and transfection was performed as described by the nctanufacturer's instructions. B7-2 expression was assessed by irzzznunofluorescence 72 h post transfection. The linearized pRc/CMV-B7-2 cDNA
(20 g or 30 g digested with Pvu I) was then electroporated into A20 cells in independent transfections using a Bi.oRadT"' electroporator. These cells were selected in G418 at a final concentration of 500 g/mL 48 h after transfection. Positive transfectants from bulk cultures were further cloned by limiting dilution. Generation of the A20/mIL-12 and A20/B7-1/mIL-12 variants has previously been described (Pizzoferrato et al., 1997).
fnxmunostaining ancl FACS analysis. Same as for IL12 and/or B7-1.
Animal Studies. For the first tumourigenicity study, 105 live A20/pRc/pac vector control, A20/$7-2 clones 3 and 5, A20/rraIL-12, or a combination of A20/rnIL-12 and A20/B7-2 variants in log phase were harvested, washed, and resuspended in 0.2 mL
RPMI 1640 medium lacking any supplements. Animals were injected subcutaneously into the right flank and monitored for tumour incidence and survival. Sixty-five days post injection, surviviria animals were challenged on the opposite flank with 105 live A20 parental cells. In the second turn,ozigenicity experiment, 105 A20/pRc/CMV
vector control, A20B7-2 clones X and 5 and cells derived from A20/$7-2 bulk cultures were inoculated into the right flank of BALB/c mice. Fifty-six days post immunization, surviving animals were challenged with the parental A20 cells on the opposite flank.
Naive aninSats injected with the parental line were used as controls. In the third turnourigenicity experiment, BALB/c rni.ce were inoculated with 105 A20/pRc/CW
vector control cells, A20/B7-2 clone 3, or A20/B7- I variants and monitored for survival.
For established disease experiments, 105 A20 cells were inoculated into the left #lank of BALB/c mice and 24 h later, 105 cells of each variant were injected into the right ,Oank.
For all the above experiments, 10 ear-tagged animals were used for each experimental group. For injection into nude animals, 105 A20/B7-1, A20/B7-2 clone 3, or A20/pRc/CMV were inoculated into BALB/c nu/nu mice and survival was monitored.
Five animals were used for each group. All animals were euthanized when tumours reached 2 cm in diameter or began to ulcerate.
Jam test. The Jam CTL test was performed as described by Matzinger (Matzinger et al., 1991). Briefly, spleens were dissected and single cell suspensions were made from BALB/c mice immunized with 105 A20/pRc/pac vector control, A20/B7-2 clones 3 and 5, A20/mIL-12, or a combination of 105 A20/B7-2 clones 3 or 5 and 105 A20/mIL-variants on the right flank. These responder cells were incubated with irradiated A20 parental cells. Viable effector cells were then incubated with 3H-thymidine (Amersham) labeled A20 target cells at different E:T ratios in a 96 well plate (Nunc, cat.l-63320) for 4 h at 37 C. After 4 h, the cells were harvested with a cell harvester and counted by scintillation counting. % specific killing was calculated as follows:
(Spontaneous cpm -Experimental cpm)/(Spontaneous cpm) x 100. The standard deviation of the mean did not exceed 10 %.
As controls, C57BL/6 mouse spleens were used as responders and irradiated BALB/c mouse spleens were used as stimulators in an allogeneic mixed lymphocyte reaction.
Naive BALB/c mouse spleens and spleens derived from mice immunized with the vector control variant incubated with the irradiated parental A20 cell line were used as negative controls. Cpm were taken of target cells alone at to and t4 to confirm that spontaneous cell death was not occurring. Also, other studies were conducted in which viable effector cells incubated at different E:T ratios with labeled targets were harvested at to to confirm that the labeled target cells were not spontaneously dying.
Depletion of T cell Subsets. Same as for IL 12 and/or B7-1.
Preparation of NK effectors from immunized animals: Spleens were pressed through a wire mesh screen with a disposable syringe plunger into complete medium (CM) which is a-MEM (Gibco, BRL, Burlington, Ontario, Canada) containing 10% FCS (Gibco, BRL), 50 M 2-ME and 10 mM HEPES. Released cells were layered over 5 ml of 6%
BSA in PBS and centrifuged to remove cell debris. The splenocytes were loaded onto a nylon wool column (Julius et al., 1973) and incubated for 1 hr at 37 C. NK
cells were enriched (69-77%) in the nylon wool non-adherent fraction, and were used as effectors in a 51Cr-release cytotoxicity assay. Anti-CD3-phycoerythrin conjugated (clone 29B) purchased from Sigma (St.Louis, MO) and DX5-FITC mAb which is a novel pan-NK
cell marker that binds an as yet unknown surface molecule on NK cells from mice of all strains tested to date, purchased from Pharmingen (San Diego, CA) were used to assess the purity of the isolated NK population.
Preparation of lymphokine activated killer culture (LAK): splenocytes from BALB/c nulnu mice were suspended in 10 ml CM, supplemented with 500 U/ml murine rIL-2 obtained from a cell line transfected with the mouse IL-2 gene (Karasuyama et al., 1989). The cells were incubated at 37 C, 10% C02 /air atmosphere for 2 days.
On the day of experiment, both adherent cells and nonadherent cells were collected, washed and resuspended in 5 ml CM, underlaid with 5 ml lympholyte-M (Cedarlane Laboratories, Hornby, Ontario), and centrifuged at 500 g for 20 min to remove dead cells.
The viable cells were washed, resuspended in CM, and used as effectors in 51 Cr-release cytotoxicity assay.
Preparation of NK targets: YAC-1 (murine lymphoma, TIB 160) and P815 (murine mastocytoma, TIB64) were obtained from the ATCC. They were grown in CM and passed three times a week. A20 and A20 transfectants were prepared as described previously.
51Cr release cytotoxicity assay: Target cells (1 x 107) were labeled with 0.4 mCi of sodium 51 Cr-chromate (Dupont Chemicals, Mississauga, Ontario, Canada) for 1 hr.
After three washes in CM, the target cells were added to 96-well V-bottomed microtiter plates in 100 l aliquots. LAK effectors (in 100 l ) were added to the radiolabeled targets at various E/T ratios in replicates. The plates were centrifuged at 400 rpm for 5 min. After a 4 hr-incubation at 37 C, the plates were centrifuged at 700 rpm and the supernatants were harvested for determination of radioactivity levels in a gamma counter.
Specific lysis was calculated as described elsewhere (Kung and Miller, 1997).
Statistical Analysis. Kaplan Meier survival curves were compared using the log rank test in the SPSS 6.1 Macintosh Version statistical package. Differences were considered statistically significant when p<0.05.
Results for B7-2 Generation of A20 variants. The A20 murine B-cell lymphoma is IgM-, IgG+, IgA-, Ia+, Fc receptor+, and complement receptor- (Kim et al., 1979). A20 also expresses both MHC class I and class II molecules, and intermediate levels of B7-2 as determined by indirect immunofluorescence staining (Fig.8). B7-1 protein expression is undetectable.
Variants expressing B7-1, B7-1/mIL-12, and mIL- 12 were described previously (Pizzoferrato et al., 1997). The B7-1 and B7-1/mIL-12 variants had comparable levels of 137-1 protein expression as measured by immunofluorescence staining (Figure 1). Vector control variants were established by transfection of pRc/CMV and retroviral infection of MSCV/pac (Pizzoferrato et al., 1997). Variants expressing B7-2 at levels higher than that seen in the parental A20 cell line were generated by transfection with the pRc/CMV/B7-2 plasmid. All positive clones were selected in G418 and further subcloned by limiting dilution.
Preliminary allogeneic mixed lymphocyte reactions were conducted using purified CD4+ T cells from lymph nodes derived from C57BL/6 mice. These T cells were stimulated with the A20-parental, A20/pRc/CMV, and A20/B7-2 bulk clone. IL-4 secretion was assayed using the CT.4S cell line and has previously been described (Pizzoferrato et al., 1997). Both the A20-parental and A20/pRc/CMV lines stimulated CD4+ T cells to secrete IL-4 resulting in CT.4S proliferation. The A20/B7-2 variant expressing more than two fold the level of B7-2 resulted in a statistically significant increase in CT.4S proliferation (data not shown). These data suggest that the endogenous B7-2 expressed by the A20 parental cell line is functional and that even slight increases in B7-2 expression could enhance T cell activation in vitro.
The clones selected for the in vivo studies (A20/B7-2-1, A20/B7-2-3, A20/B7-2-5) expressed approximately 2.5X more B7-2 protein expression than the parental A20 clone as indicated by immunofluorescence staining. Immunofluorescence staining indicated that expression of surface molecules MHC class I and II was unaltered in these clones (Fig.8). The phenotypic profile of these variants did not change upon reassessment after several weeks in culture. Moreover, the doubling rates of the A20-parental and variant lines were not significantly different (data not shown).
A20 Transfectants Expressing B7-2, and/or m1L-12 are Rejected By Syngeneic BALB/c mice and induce protective systemic anti-tumour immunity.
105 live tumour cell variants including A20B7-2-3, A20/B7-2-5, A.20/mIL-12, or a combination of A20/B7-2 transfectants clone 3 or 5, and A20/mIL- 12 transfectants were injected subcutaneously into 10 mice each. Animals inoculated with the vector control or parental A201ine developed progressive tumours and most of these mice were euthanized by day thirty-six (Fig.9). The A20/mIL- 12 transfectant was completely rejected by BALB/c mice. Of the animals inoculated with either of the two A20/B7-2 clones, only two animals developed a tumour and this difference is statistically insignificant when compared to the survival curves of the IL-12 variant (p>0.05) (Fig.9).
Coinjection of the A20/B7-2 and A20/mIL-12 variants did not result in a statistically significant improval in survival when compared to survival of mice injected with either one of these variant lines. 65 days post the initial inoculation the surviving animals were challenged with 105 A20 parental cells on the opposite flank. By day forty-five, 6 out of the 9 previously unimmunized control animals had developed tumours and were euthanized. One animal from the group initially immunized with the A20/B7-2 clone 5 group developed a tumour and was euthanized on day thirty-five. All other animals inoculated with the transfectants and challenged with the unmodified parental cell line did not develop tumours (data not shown).
In a repeat of this experiment, mice were injected with 105 cells from either two independent A20/B7-2 bulk cultures, A20/B7-2 clone 1 or 5, or A20/pRc/CMV
vector control variants. Only animals inoculated with the A20/pRc/CMV vector transfectant developed progressive tumours. Fifty-six days post initial inoculation protected animals were challenged with parental cells on the opposite side. By day thirty-five post challenge, only one mouse initially immunized with the A20/B7-2-5 variant clone had developed a tumour. In contrast, only 20% of the animals in the control nonimmunized group were still alive by day forty-three (data not shown). Tumours did not develop in any of the other animals (data not shown).
In both of the above challenge experiments, mice inoculated with the A20/B7-2 variants had statistically significant increases in survival over animals immunized with the parental or vector control A20 cell lines. Also, there was no difference in survival when the animals were inoculated with the A20/B7-2 variants alone or with a combination of A20/B7-2 and the A20/mIL-12 variants. Collectively, these tumourigenicity studies suggest that tumour cells genetically modified with B7-2 induce anti-tumour responses and that these are as potent in inducing anti-tumour immunity as tumour cells genetically modified with IL-12.
B7-2 is more effective than B7-1 in the A20 B cell lymphoma murine model system.
To compare the effectiveness of A20/B7-1 and A20/B7-2 in a vaccination animal tumour model, 105 A20/B7-1, A20/B7-2 clone 3, and A20/pRc/CMV variants were inoculated into 10 animals each. The animals inoculated with the B7-I or vector control variant developed progressive tumours and 90 % were euthanized by day thirty-eight. In contrast, all animals inoculated with the B7-2 clone remained tumour free with a statistically significant increase in survival when compared pairwise with the animals injected with the A20/pRc/CMV vector control (p<0.0005) or animals inoculated with the A20/B7-1 variant (p<0.0005) (Fig. 10). Statistically significant differences in survival were not observed between animals injected with the vector control or A20/B7-1 variant.
Reduction ofB7-2 Expression in a mouse immunized with A20/B7-2. Only 4 animals of a total of 30 mice inoculated with the A20/B7-2-5 clone developed progressive tumours. One of these tumours was dissected and immunofluorescence staining performed. Figure 11 demonstrates that the level of B7-2 expressiori in the tumour cells is a little different from the endogenous level expressed by the parental cells, i.e., the level of B7-2 had decreased from that seen in the clone A20/B7-2-5. Although all the clones employed in the above experiments were cloned by limiting dilution it is unclear whether these tumour cells were wild-type contaminants in the A20/B7-2-5 inoculum or whether B7-2 down regulation occurred in vivo. These results suggest that a certain threshold level of B7-2 is necessary to mediate an anti-tumour imniune response and that in some situations tumour cells with lower levels of B7-2 expression may escape this anti-tumour response.
B7-2 modified tumour cells enhance survival in a minimal disease tumour model.
To mimic the clinical setting of established disease, animals were injected with parental cells on the left flank and 24 h later, animals were inoculated with 105 tumour variants on the opposite (i.e., right ) flank. Because profound anti-tumour immune responses were observed when A20 had been genetically modified with B7-1/mIL-12, comparisons of these variants in an established disease model were conducted.
Sixty-eight days post injection of the variant lines, 20% (2/10) of the mice remained alive in the vector control groups, 40 % survived in the A20/B7-l group, 30% survived in the mIL-12 group, 40 % survived in the B7-1 /mIL-12 group, and 70 % survived in the B7-2 group.
Moreover, the surviving animals injected with the A20/B7-2 variant were tumour free and had statistical increases in survival when compared to either the A20/pRc/CMV
(p<0.006) or A20/pRc/pac (p<0.02 - vector control groups (Fig. 12). In addition, a statistically significant increase in survival (p<0.072) was approached in favour of the B7-2 group when the A20/mIL-12 group was compared to the animals injected with the A20/B7-2 variant. Although the survival curves were superior, statistical differences in survival were not observed when the A20/B7-2 animals were compared with the 1 /mIL-12 variant (p>0.1).
In a more stringent experiment, we inoculated 105 A20 parental cells in the left flank and four days later, these mice were inoculated in the opposite flank with either 105 A20/pRc/CMV vector control, A20/B7-2-5 clone, or A20/B7-2 bulk variant cell lines. In this experiment, the protective effect of B7-2 was not observed. The mice injected with the A20/pRc/CMV variant developed tumours on both flanks whereas the mice injected with the A20/B7-2 variants developed tumours only on the left flank. The statistical analyses indicated that there was no statistical increase in survival in any of the three groups (data not shown).
Taken together, in this minimal disease model, animals injected with A20/B7-2 variant demonstrate anti-tumour immune responses, that are just as potent or greater than anti-tumour responses induced with the A20/B 7-1 /mIL-12 or A20/mIL-12 groups.
However, there are limitations on the potency of this effect since effective anti-tumour immunity can only be achieved in a limited disease state.
Mechanisms by which A20/B7-2 mediates anti-tumour immune responses. To gain insight into the mechanism responsible for the observed anti-tumour activity elicited by tumour cells engineered with B7-2, T cell deficient BALB/c nu/nu mice were inoculated with 105 A20/pRc/CMV, A20/B7-1, or A20/B7-2 variants. By day twenty-nine post injection all animals were euthanized with tumours approximating 2 cm in diameter (Fig.
13). Unlike the results seen in immunocompetent mice no statistical differences in survival were seen between the animals injected with the B7-1 or B7-2 transfectants or the vector control (p>0.08). these data suggest that T cells are important in mediating the anti-tumour response produced by the variant expressing higher levels of B7-2.
Since nude mice contain normal or even elevated levels of NK cells, these data also imply that NK cells play a minimal role.
In addition, CTZ, assays as measured by DNA fragmentation were performed usinb animals that had been immunized with the A20/pRe/pac vector control, A20/B7-2 clones 3 or 5, or the A20/mIL-12 variant lines. Animals were also inununized with combinations of A20/B7,2 and A20/miL-12 to detezxnine if the IL- 12 would synergize with the B7-2 in the inoculum to elali:ance CTL activitv. As shown +ri FigurP
iA, at -A-- === --- ---=
effector to target ratio of 40_ 1, CTL lytic activity frorn animals injected with either the A20B7-2-3 or A20/B7-2-5 clone was 78.3% and 77.2% respectively which was comparable or greater to the CTL activity seen from rnice inimunized with the variants. Coin,jection with the IL-12 variant did not augment the activity seen with the B7-2 variants.
To determine which T cell subset was predominantly mediating the observed anti-tumour imrrxune response in vivo, CD41 and CD8' T cells wcre depleted using mAbs.
By day nineteen, 60% of the mice depleted of CD8+ T cells and inoculated with the A20B7-2 variant had developed palpable tumours (Fig. 15), while animals remained tumour $ee in the CD4+ depleted group (data not shown). Interestingly, when CD8} T
cell depletion was terminated on day thirty-two, the tumours which had developed in mice inoculated with the A20/87-2 variant began to regress and by day thirty-seven only one mouse had a palpable tum.our remaining (Fig. 15). Taken together the depletiozi data and the CTL lysis results suggest that CD8+ T cells play a major role in mediating the aaxti-tumour immune response.
Both CD8+or CD41 T cell depleted mice inoculated with the A20/B7-1/mIL-12 variant were still able to reject this variant confirming our previous experiments (Pizzoferrato et al., 1997) and suggesting that the meclianism of anti-tumour immurnity mediated via these tumour cells is multifactorial.
Lymphokine activated killer cell (LAK) cultures were prepared by incubating BALB/c nu/nu splenocytes in media containing IL-2 for 48 h to determine whether natural killer cells could be functioning to mediate anti-tumour immune responses observed with the A20/B7-2, A20/mIL-12 or A20/B7-1 /mIL- 12 transfectants. Figure 16 demonstrates that the parental A20 cell line is a target for lysis by LAK cells. Differences in killing by these LAK cells were not observed between the parental and transfectant lines.
However, when the A20 cell variants were injected intravenously into BALB/c nu/nu mice and NK
cells were isolated from immunized mice, differences in killing capacity were detected.
Figure 17 indicates that the intravenously injected IL- 12 variants were capable of augmenting the natural killer cell killing of A20 parental cells when compared to the A20/B7-2 and vector control variants. These data illustrate that although the A20 is an effective target for killing by natural killer cells, the IL- 12 variants can augment the killing capacity. Moreover, although the A20/B7-2 variant elicits potent anti-tumour immune responses, natural killer cells play a minimal role in the observed augmented immune response.
Discussion for B7-2 Effective immune responses in murine tumour models have been elicited by introducing the costimulatory molecules, B7-1 or B7-2 (Chen et al., 1992;
Townsend et al., 1993; Yang et al., 1995; Gajewski et al., 1996; Martin-Fontecha et al., 1996). The relative effectiveness of B7-1 or B7-2 in generating anti-tumour immunity may depend upon the inherent immunogenicity of the tumour. In recent studies, B7-2 modified tumour cells induced superior anti-tumour responses in non-immunogenic tumours while B7-1 was just as effective as B7-2 in immunogenic tumours (Martin-Fontecha et al., 1996). In contrast, Matulonis et al., reported more profound anti-tumour effects when B7-1 was introduced into a non-immunogenic murine myeloid leukemia model (Matulonis et al., 1996).
Unlike most solid tumours which do not express B7-1 or B7-2, immunostaining demonstrates that B cell malignancies express the costimulatory molecule B7-2 (Schultze et al., 1995; Xerri et al., 1997; Van Gool et al., 1997). Despite the expression of this potent costimulator as well as MHC I and MHC II, freshly isolated patient samples of follicular lymphoma cannot generate efficient T cell responses (Schultze et al., 1995).
Only when stimulated via the CD40-L can these cells stimulate T cells in an allo MLR
(Schultze et al., 1995). Like most human B cell lymphomas, the non-immunogenic inurine B cell lymphoma does not express B7-1, but does express moderate levels of B7-2. Although the presence of B7-1 could indeed stimulate allogeneic CD4+ T
cells to secrete both IL-2 and IL-4, a profound in vivo anti-tumour immune response was not observed with B7-1 modified lymphoma cells (Pizzoferrato et al., 1997).
Despite the presence of moderate endogenous levels of B7-2 in the A20 cell line, only minimal levels of IL-4 are secreted from allogeneic T cells and 11-4 secretion is enhanced when B7-2 expression is augmented, suggesting that there may be a threshold of B7-2 expression required to induce T cell activation (Pizzoferrato et al., 1997). It is thus hypothesized that enhancement of B7-2 expression may lead to enhanced T cell activation and perhaps anti-tumour immune responses in a murine model of B cell lymphorna.
Indeed, when tumourigenicity studies were conducted with various A20 clones expressing only 2.5X the level of B7-2 than the parental cell line, immunogenicity was significantly augmented and these variants were rejected in syngene:ic hosts.
Moreover, the protected animals rejected challenge with the parental, unmodified cell line.
Interestingly, tumour escape variants were observed from the A20/B7-2 clone which had lost expression of B7-2 to the level seen in the parental clone. This observation is consistent with the idea that a critical threshold of B7-2 may be necessary to elicit anti-tumour immunity in tumour cells expressing endogenous B7-2. Recently, it has been shown that the effectiveness of low dose L-PAM chemotherapy is associated with the upregulation of B7-2 expression in the draining lymph nodes of MOPC-315 plasmacytoma tumour bearers. Since MOPC-315 cells from mice treated with L-PAM
express higher levels of B7-2 than B7-1, it is suggested that this higher expression may contribute to the importance of B7-2 in the MOPC-315 tumour system (Mokyr et al., 1998).
Strikingly, in a minimal disease tumour model, in which the parental line was inoculated 24 hr prior to vaccination with each of the variant lines, only animals treated with the A20/B7-2 clone had a statistically significant increase in survival when compared to the vector controls at the end of a 68 day time interval. In addition, 70% of these animals remained tumour free.
When the A20/B7-2 variants were inoculated 4 days post inoculation of the parental cell line, the anti-tumour protection was lost. A number of factors including the rapidity of tumour growth, or peripheral tolerance to tumour antigens may explain this observation. It has recently been shown that clonotypic CD4+ T cells initially expand a.nd lose their naive phenotype in mice injected with both A20/influenza hemagglutinin (HA) tumour cells and adoptively transferred anti-HA/I-Ed transgenic T cells.
These HA-specific T cells, however, have diminished proliferative responses to HA
peptide in vitro (Staveley-O'Carroll et al., 1998). This unresponsiveness was observed as early as 6 days post T cell transfer. Peripheral tolerance may occur in our mociel system as well. If this hypothesis is indeed correct, then subsequent restimulation with A20/B7-2 higher expressing variant tumour cell lines would result in similar unresponsiveness and these wuznals would be unable to mount effective anti-tumour responses. Moreover, T
cell anergy txtay result from low level 137 expression and selective binding to the negative regulator of iinmune responses, CTLA-4 (Perez et al., 1997).
In the first part of this description, it was shown that tumour cells modified with IL-12 in addition to B7-1 wm more effective in inducing anti-tClnl,our immunity than tuinoAr cells modified with either molecule alone. To investigate whether similar synergy exists between B7-2 and IL-12, mice were coinjected with both A20/B7-2 and A20/mIL-12 clones. In contrast to B7-1 and IL-12, synergy between B7-2 and IL-12 was not demonstrated in this murine model since the survival of animals inoculated with the $7-2 variants was not statistically different from that of mice coinjected with the and A20/mrL-12 variant clones.
Although a statistical difference was not noted between the A20/B7-2 and the A20/mIL-12 group in this minimal disease model, the survival of animals inoculated with A20/B7-2 approached statistical significance when compared to the A20/mIL-12 group.
Thi.s subgests that enhanced expression of B7-2 is equaE to or cnay be more effective than IS.-12 in generating anti-tumour irn.mune responses.
In several tumour models, enhanced anti-turnour immunitv elicited by B7-1 or B7-2 is primarily T cel.l mediated, although B7-1 has also been shown to enhance NK
lysis (Chambers et aL, 1996). While anRi-tumour immunity generated by B7-1 is primarily CD8+ T cell mediated (Townsend et al., 1993; Matulonis et al_; 1996), studies have demornstrated that CD4+ and/or CDS+ T cells are responsible for anti-tumour responses elicited by B7-2 (Martin.-p'ontecha et al., 1996; Matul.onis et aI., 1996). In this mtirine model of B cell lymphoma, the protection observed with the A20/B7-2 variants is inediated primarily by CD8+ T lymphocytes. Anti-tumour immunity was lost when A20/B7-2 variants were inoculated in nu/nu mice and more specifically in CD8 depleted but not CD4 depleted mice. In addition, enhanced CTL lysis as measured by DNA
fragmentation was seen in animals immunized with the B7-2 clones.
The mechanism of anti-tumour activity mediated by B7-2 modified A20 cells is different from that mediated by IL-12 or a combination of B7-1 and IL-12 modified lymphoma variants.
B7-1 can act as a triggering signal for NK lysis and may play a role in anti-tumour responses elicited by B7-1 transfected tumour cell lines (Yeh el al., 1995;
Wu. T.C. et al., 1995; Cavallo et al., 1995; Geldhof et al., 1995). For example, the 'T cell lymphoma, EL4, when transfected with B7-1, is efficiently killed by lymphokine-activated killer cells (LAK cells) (Chambers et al., 1996). In order to address whether LAK
cells or NK
cells could mediate the observed A20/B7-2 anti-tumour responses, LAK and NK
assays were performed with each of the variant cell lines. Lysis of A20 parental and variant lines by in vitro generated LAK cells was comparable. When intravenously injected into nu/nu mice, however, NK cells derived from mice immunized with the IL-12 or B7-12 variants were capable of augmented NK killing of the A20 target. This suggests that NK cells play a more important role in mediating the observed anti-tumour response in mice immunized with the A20/mIL- 12 or A20/B7-1/mIL-12 variant lines than with the B7-2 modified variants. This is consistent with studies demonstrating the effectiveness of IL-12 in enhancing NK cell mediated anti-tumour activity (Tahara et al., 1995).
Unlike Chambers et al. whose studies showed triggering of mouse LAK cells by (Chambers et al., 1996), these data do not show enhancement of LAK cell killing through the introduction of B7-1 or B7-2 into the A20 murine B cell lymphoma cell line. These data show that A20 is an NK-sensitive target and any increase in costimulation may not further enhance its susceptibility to NK mediated lysis. Moreover, despite the expression of MHC class I which acts as an NK inhibitory signal (Ljunggren et al., 1990;
Karre et al., 1995), the A20 parental and variant cell lines are sensitive to lysis by NK cells.
However, progressive tumours develop in mice inoculated with the A20 parental or A20/pRc/pac vector control variants suggesting that NK cell activity alone is not potent enough to prevent tumour outgrowth.
Although evidence indicates that B7-1 and B7-2 may have distinct functional roles, the relative effectiveness of either molecule in immunotherapeutic strategies aimed at treating cancer patients remains unclear. It is perhaps not surprising that different anti-tumour activity may be mediated by B7-1 or B7-2 costimulation. These molecules may differentially influence the immune response. For example, in vitro stimulation assays and evidence obtained by the murine model of experimental allergic encephalomyelitis (EAE) suggest that B7-2 stimulates naive CD4+ T cells to secrete IL-4, hence, skewing the immune response towards a ThO/Th2 phenotype (Kuchroo et al., 1995; Freeman et al., 1995). In contrast, B7-1 allows Th precursor cells to differentiate towards a Thi phenotype. These data indicate that introduction of B7-2 into the non-immunogenic B
cell lymphoma, A20, can enhance CTL activity and allow for profound anti-tumour immune responses. Taken together, anti-tumour immunity generated by B7-1 or B7-may depend upon both the immunogenicity of the tumour cell line as well as the cytokine environments induced by these costimulatory molecules.
This is particularly relevant to human B cell lymphomas which generally express moderate levels of B7-2 yet act as poor APCs (Schultze et al., 1995). This murine model niimics this phenomenon and demonstrates that a critical threshold of costimulatory activity may be necessary to activate "T cells and generate anti-tumour immune responses in B cell malignancies. These results may have important implications for immunogene therapy approaches aimed at treating B cell lymphomas.
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Xerri et al., have also shown that the majority of follicular and diffuse large B cell lymphomas expressed both B7-1 (32/44 samples) and B7-2 (39/44 samples) (Xerri et al., 1997). In addition, it has been shown that expression of B7-1 and B7-2 was restricted to the malignant B cells of the germinal center (Dorfman et al., 1997). I liave shown expression of B7-2, but no expression of B7-1 in patient samples as well as B cell lines (data not shown).
Moreover, recent in vitro data demonstrate that low levels of B7 expression in human follicular lymphoma samples was unable to induce profound T cell proliferation, but was sufficient to prevent the induction of alloantigen-specific anergy. Activation of these malignant B cells by CD40 stimulation using CD40L transfectants upregulated B7-1 and B7-2 expression resulting in proliferation of allogeneic T cells (Dorfman et al., 1997). It was suggested that the quantitative level of B7 expression determines T cell responsiveness.
Because the A20 murine B cell lymphoma expresses low endogenous levels of B7-2, it was reasoned that this cell line would be ideal to investigate how modulation of levels of endogenous expression affects immunogenicity. A20 is non-immunogenic in that immunization with 106 irradiated cells or 105 live cells does not protect animals from challenge with live parental unmodified cells (Levitsky et al., 1996;
Pizzoferrato et al., 1997). This tumour cell line is tumourigenic despite moderate levels of B7-2 expression and high levels of both MHC I and II. Previous studies demonstrated that the immunogenicity of the A20 B cell lymphoma is enhanced with the introduction of J
and more potent responses are elicited with the combination of B7-1 and IL- 12 (Pizzoferrato et al., 1997).
In this study, the murine A20 cell line genetically engineered to express B7-2 at levels greater than the endogenous level elicit profound in vivo anti-tumour immunity. While animals injected with B7-1 variants succumb t.o tumour, animals inoculated with variants expressing greater levels of endogenous 137-2 reject tumour grow-th and are protected against subsequent challenge with unmodified parental cells. Moreover, CTLs are generated in protected animals. The potency of the anti-tumour response is equal to that observed with A20 variants genetically modified to express IL- 12 or both B7-1 and IL-12. In addition, vaccination of tumour bearing animals demonstrates the therapeutic potential of B7-2 in preventing tumour growth. Collectively, this murine model suggests that the level of B7-2 expression determines in vivo anti-tumour responsiveness. Note that B7-2 is already present on A20. Thus, these results imply that modestly increasing its level of expression is sufficient to change a non-immunogenic tumour into an immunogenic tumour.
5a In accordance with an aspect of the present invention, there is provided an in vitro or ex vivo method of enhancing immunity against hematopoietic tumours in a mammal which has a hematopoietic tumour, said method comprising the steps of:
genetically modifying hematopoietic tumour cells from the same or a different mammal to express a cytokine and costimulatory molecule, in the mammal, wherein said cytokine and costimulatory molecule is 1L-12 in combination with B7-l, and expressing said cytokine and costimulatory molecule in the presence of cells from said hematopoietic tumour, or, genetically modifying hematopoietic tumour cells from the same or a different mammal to express a costimulatory molecule, in the mammal, wherein said costimulatory molecule is B7-2, and expressing said costimulatory molecule in the presence of cells from said hematopoietic tumour.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the step of genetically modifying further comprises the step of genetically modifying said hematopoietic tumour cells using at least one vector construct which directs the expression of a DNA sequence, said at least one vector construct comprising:
IL-12 gene in combination with B7-2 gene; IL-12 gene in combination with B7-1 gene; B7-1 gene alone ; B7-2 gene alone; or IL-12 gene in combination with B7-1 gene in combination with B7-2 gene, to form genetically modified hematopoietic tumour cells, wherein said combination may be on one vector or on more than one vector.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the at least one vector construct comprises a polynucleotide vector.
5b In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the at least one vector construct comprises a combination of a virus and a plasmid; or a virus and a polynucleotide; or a plasmid and a polynucleotide; or a virus and a plasmid and a polynucleotide.
In accordance with another aspect of the present invention, there is provided the tnethod of the present invention wherein the at least one vector construct comprises a virus.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the virus is fowlpox.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the virus is canarypox.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the virus is adenovirus.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the virus is vaccinia virus.
In accordance with another aspect of the present invention, there is provided the method of the present invention whereiin the virus is swine pox virus or polio virus.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the virus is a retrovirus.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the at least one vector construct comprises a plasmid.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the plasmid is a DNA plasmid comprising a promoter which allows for the expression of the gene or combination of genes.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the plasmid is pRc/CMV.
5c In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the hematopoietic tumour is a leukemia, lymphoma, or myeloma.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the leukemia is acute lymphoblastic leukemia (ALL), acute myelocytic leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL) or juvenile myelo-monocytic leukemia (JMML).
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the lymphoma is B-Cell Burkitt's lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Ki-1 positive anaplastic large cell lymphoma, T cell lymphoma or histiocytic lymphoma.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the myeloma is a multiple myeloma.
In accordance with another aspect ofthe present invention, there is provided the method of the present invention wherein the genetically modified tumour cells are present at the same site as a tumour burden.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the genetically modified tumour cells are present at a site which is different frorii, a tumour burden.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the genetically modified tumour cells are present at a site which is distant from a tumour burden.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein said tumour burden is defined as a minimal disease state.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the genetically modified tumour cells are present in proximity to the hematopoietic tumour.
5d In accordance with another aspect of the present invention, there is provided an in vitro or ex vivo method of enhancing immunity against hematopoietic tumours in a mammal which has a hematopoietic tumour, said method comprising the steps of:
genetically modifying hematopoietic tumour cells from the same or a different mammal to express a cytokine and costimulatory molecule, in the mammal, wherein said cytokine and costimulatory molecule is IL-12 in combination with B7-1, and expressing said cytokine and costimulatory molecule, in vitro or ex vivo in the presence of immune cells, said immune cells comprising antigen presenting cells, T cells, and/or NK cells, in the presence of naturally occurring mammalian hematopoietic tumour cells, or genetically modifying hematopoietic tumour cells from the same or a different mammal to express a costimulatory molecule, in the mammal, wherein said costimulatory molecule is B7-2, and expressing said costimulatory molecule, in vitro or ex vivo, in the presence of immune cells, said immune cells comprising antigen presenting cells, T cells, and/or NK cells, in the presence of naturally occurring mammalian hema.topoietic tumour cells, and waiting until lysis or activation of cell death processes of said naturally occurring mammalian hematopoietic tumour cells occurs.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the expression of cytokine and costimulatory molecule in vitro or ex vivo comprises introducing at least one vector construct which directs the expression of a DNA sequence, said at least one vector construct comprising:
IL-12 gene in combination with B7-2 gene; IL-12 gene in combination with B7-1 gene; B7-l gene alone ; and 87-2 gene alone; or IL-12 gene in combination with B7-1 gene in combination with B7-2 gene, 5e into the vicinity of the hematopoietic tumour, wherein said combination may be on one vector or on more than one vector.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the step of genetically modifying further comprises the step of genetically modifying said hematopoietic tumour cells using at least one vector construct which directs the expression of a DNA sequence, said at least one vector construct comprising:
IL-12 gene in combination with B7-2 gene; IL-12 gene in combination with B7-1 gene; B7-1 gene alone ; B7-2 gene alone; or IL-12 gene in combination with B7-1 gene in combination with B7-2 gene, to form genetically modified hematopoietic tumour cells, wherein said combination may be on one vector or on more than one vector.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the at least one vector construct comprises a polynucleotide vector.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the at least one vector construct comprises a combination of a virus and a plasmid; or a virus and a polynucleotide; or a plasmid and a polynucleotide; or a virus and a plasmid and a polynucleotide.
In accordance with another aspect of the present invention, there is provided the nlethod of the present invention wherein the at least one vector construct comprises a virus.
In accordance with another aspect of the present invention, there is provided the niethod of the present invention wherein the virus is fowlpox.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the virus is canarypox.
5f In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the virus is adenovirus.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the virus is vaccinia virus.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the virus is swine pox or polio virus.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the virus is a retrovirus.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the at least one vector construct comprises a plasmid vector.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the plasmid is a DNA plasmid comprising a promoter which allows for the expression of the gene or combination of genes.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the plasmid is pRc/CMV.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the hematopoietic tumour is a leukemia, lymphoma, or myeloma.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the leukemia is acute lymphoblastic leukemia (ALL), acute myelocytic leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL) or juvenile myelo-monocytic leukemia (JMML).
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the lymphoma is B-Cell Burkitt's lymphoma, Hodgkin's lymphomas, non-Hodgkin's lymphomas, Ki-l positive anaplastic large cell lymphomas, T cell lymphomas or histiocytic lymphomas.
5g In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the myeloma is a multiple myeloma.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the genetically modified tumour cells are present at the same site as a tumour burden.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein the genetically modified tumour cells are present at a site which is different from a tumour burden.
In accordance with another aspect of the present invention, there is provided the method of the present invention whereiin the genetically modified tumour cells are present at a site which is distant from a tumour burden.
In accordance with another aspect of the present invention, there is provided the method of the present invention wherein said tumour burden is defined as minimal disease state.
In accordance with another aspect of the present invention, there is provided a vaccine for enhancing immunity to hematopoietic tumours in a mammal which has a heniatopoietic tumour, said vaccine comprising genetically modified hematopoietic tumour cells of a mammal, wherein said tumour cells are genetically modified to express a cytokine and costimulatory molecule wherein said cytokine and costimulatory molecule is IL-12 in combination with B7-l, or wherein said tumour cells are genetically modified to express a costimulatory molecule wherein said costimulatory molecule is B7-2.
5h In accordance with another aspect of the present invention, there is provided the vaccine wherein the vaccine comprises live, replicating cells.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the vaccine comprises non-replicating cells.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the genetically modified hematopoietic tumour cells further comprise hematopoietic tumour cells transduced, transfected or infected using at least one vector construct which directs the expression of a DNA sequence, said at least one vector construct comprising:
IL-l2 gene in combination with B7-2 gene; IL-12 gene in combination with B7-1 gene; B7-I gene alone ; B7-2 gene alone; or IL-12 gene in combination with B7-l gene in combination with B7-2 gene, wherein said combination may be on one vector or on more than one vector.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the at least one vector construct comprises a polynucleotide vector.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the at least one vector construct comprises a combination of a virus and a plasmid; or a virus and a polynucleotide; or a plasmid and a polynucleotide; or a virus and a plasmid and a polynucleotide.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the at least one vector construct comprises a virus.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the virus is fowlpox.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the virus is canarypox.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the virus is adenovirus.
5i In accordance with another aspect of the present invention, there is provided the vaccine wherein the virus is vaccinia virus.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the virus is swine pox virus or polio virus.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the virus is a retrovirus.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the at least one vector construct comprises a plasmid.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the plasmid is a DNA plasmid comprising a promoter which allows for the expression of the gene or combination of genes.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the plasmid is pRc/CMV.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the hematopoietic turnour is a leukemia, lymphoma, or myeloma.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the leukemia is acute lymphoblastic leukemia (ALL), acute myelocytic leukemia (AML), chronic rnyeloid leukemia (CML), chronic lymphocytic leukemia (CLL) orjuvenile myelo-monocytic leukemia (JMML).
In accordance with another aspect of the present invention, there is provided the vaccine wherein the lymphoma is B-Cell Burkitt's lymphoma, Hodgkin's lymphomas, non-Hodgkin's lymphomas, Ki-1 posiltive anaplastic large cell lymphomas, T
cell lymphomas or histiocytic lymphomas.
In accordance with another aspect of the present invention, there is provided the vaccine wherein the myeloma is a multiple myeloma.
In accordance with another aspect of the present invention, there is provided a pharmaceutical composition for use as a vaccine for enhancing immunity to hematopoietic tumours in a mammal which has a hematopoietic tumour, said 5j pharmaceutical composition comprising, genetically modified hematopoietic turnour cells of a mammal wherein said tumour cells are genetically modified to express a cytokine and costimulatory molecule wherein said cytokine and costimulatory molecule is IL-12 in combination with or genetically modified hematopoietic turnour cells of a mammal wherein said tumour cells are genetically modified to express a co-stimulatory molecule wherein said costimulatory molecule is B7-2;
together with a pharmaceutically acceptable carrier or diluent.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein said genetically modified hematopoietic tumour cells further comprise hematopoietic tumour cells transduced, transfected or infected using at least one vector const:ruct which directs the expression of a DNA
sequence, said at least one vector construct comprising:
IL-12 gene in combination with B7-2 gene; IL-12 gene in combination with B7-1 gene; B7-1 gene alone ; B7-2 gene alone; or IL-12 gene in combination with B7-1 gene in combination with B7-2 gene, wherein said combination may be on one vector or on more than one vector.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the at least one vector construct comprises a polynucleotide vector.
In accordance with another aspect of the present invention, there is provided the composition wherein the at least one vector construct comprises a combination of a virus and a plasmid; or a virus and a polynucleotide; or a plasmid and a polynucleotide; or a virus and a plasmid and a polynucleotide.
5k In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the at least one vector construct comprises a virus.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the virus is fowipox.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the virus is canarypox.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the virus is adenovirus.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the virus is vaccinia virus.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the virus is swine pox virus or polio virus.
In accordance with another aspect of the present invention, there is provided the composition wherein the virus is a retrovirus.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the at least one vector construct comprises a plasmid.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the plasmid is a DNA plasmid comprising a promoter which allows for the expression of the gene or combination of genes.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the plasmid is pRc/CMV.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the hematopoietic tumour is a leukemia.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the hematopoietic tumour is a lymphoma.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the hematopoietic tuniour is a myeloma.
In accordance with another aspect of the present invention, there is provided the coniposition of the present invention wherein the leukemia is acute lymphoblastic leukemia (ALL), acute myelocytic leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL) or juvenile myelo-monocytic leukemia (JMML).
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the lymphoma is B-Cell Burkitt's lymphoma, Hodgkin's lymphomas, non-Hodgkin's lymphomas, Ki-1 positive anaplastic large cell lymphomas, T cell lymphomas or histiocytic lymphomas.
In accordance with another aspect of the present invention, there is provided the composition of the present invention wherein the myeloma is a multiple myeloma.
In accordance with another aspect of the present invention, there is provided the pharmaceutical composition for use as a vaccine for reducing hematological tumour burden in a mammal.
In accordance with another aspect of the present invention, there is provided the pharmaceutical composition for use as a vaccine wherein said vaccine is suitable for administration intravenously, intradermally, orally, intranasally, subcutaneously, intranodally, intralymphatically, or intrathecally.
In accordance with another aspect of the present invention, there is provided the pharmaceutical composition for use as a vaccine wherein said vaccine is suitable for administration at the site of the hematological tumour.
In accordance with another aspect of the present invention, there is provided the pharrr-aceutical composition for use as a vaccine wherein said vaccine is suitable for administration at a site different from the hematological tumour.
5m In accordance with another aspect of the present invention, there is provided the pharmaceutical composition for use as a vaccine wherein said vaccine is suitable for administration at a site distant from the hematological tumour.
In accordance with another aspect of the present invention, there is provided the pharmaceutical composition in the preparation of a medicament or a medicine wherein said medicine or medicament is suitable for administration intravenously, intradermally, orally, intranasally, subcutaneously, intranodally, intralymphatically, or intrathecally.
In accordance with another aspect of the present invention, there is provided the pharmaceutical composition in the preparation of a medicament or a medicine wherein said medicine or medicament is suitable for administration at the site of the heniatological tumour.
In accordance with another aspect of the present invention, there is provided the pharmaceutical composition in the preparation of a medicament or a medicine wherein said medicine or nledicament is suitable for administration at a site different from the hematological tumour.
In accordance with another aspect of the present invention, there is provided the pharmaceutical composition in the preparation of a medicament or a medicine wherein said medicine or medicament is suitable for administration at a site distant from the hematological tumour.
Brief Description of the Figures Figure 1. Indirect immunofluorescence profiles of A20 parental and variant lines. A20 parental and variant lines were stained for B7-1, B7-2, and MHC class I and II
molecules.
Stippled lines represent staining with isotype controls; Solid lines represent staining with the primary mAb followed by secondary FITC goat anti-rat (B7-l and B7-2) or streptavidin (MHC I and MHC II) fluorescent conjugate. The FITC goat anti-rat conjugated antibody alone was used as a control for IgG staining.
Figure 2. Cytokine production of purified allogeneic CD4+ T cells to A20 variants.
CTLL proliferation in response to IL-2 in supematants derived from allogeneic mixed lymphocyte tumour cultures (A), CT.4S proliferation in response to IL-4 in supernatants derived from allogeneic mixed lymphocyte reactions (B), IFN-y production by CD4+ T
cells to A20 variants as assessed by ELISA (C).
Figure 3. IL-2 production is inhibited by anti-B7-1 in mixed lymphocyte tumour cultures. Monclonal antibodies 1610A1 completely inhibits IL-2 production in mixed lymphocyte cultures consisting of purified allogeneic CD4+ T cells and A20/B7-1 (A) or A20/B7-1 /mL-12(B).
Figure 4. Percent Survival of animals injected with A 20 and variant cell lines. Kaplan Meier survival statistics were performed as described in Materials and Methods. Events were recorded when animals were euthanized for tumours that had reach 2 cm in diameter or that had ulcerated. Survival for A20/B7-1, A20/mIL-12, and A20/B7-1mIL-12 are superior and statistically significant (p<0.005) when compared pairwise with the A20/pRc/CMV vector control.
Figure 5. Percent Survival of animals injected with A20 variants and A20 parental cells at different sites and at the same time. Animals were inoculated with 105 variant cells on one flank and 105 variant cells on the opposite flank. Survival, was monitored. Log rank pairwise comparison demonstrated that only survival for the A201137-1/mYL-12 treatment group was statistically significant when compared to the A20/pRc/CMV or A20/pRc/pac (p<0.02). Both the A20/[n1L-12 and A201137-1/mIL-12 treatment groups had better survival than the animals inoculated with the A20B7-1 variant and the unmodified parental cells (p<0.44).
Figure 6. Percent Survival of animals inoculated with a mixture of 10' parent and 10$
variant lines at the sarne site. Log rank pairwise comparison demonstrated that both the A20/rnIL-12 and the A20/B7-1/rcdL-12 treatment groups had better survival than the A20/pRc/CM't% treatment group (p<0.0001). The survival of the A,20/H7-1/mIL-12 treatment group was also statistically superior when compared to the A20/pRc/pac treatment group (p<0_001), the A20B7-1 group (p<0.0001), and the A20/mIL-12 treatment group (p<O.OOS).
Figure 7. Generation of CTLs in animals imrnuwxized with the A20/rn.IT; 12 or ]/mIL-12 variant lines and subsequently challenged with the A20 parental cell line_ A
syngenic mixed lymphocyte tumour culture was perfurmed against the irradiated parental line by usina spleen cells as responders from mice that had previously been immunized with the A20/nmlL-12 or A20/87-1/mYL-12 variant lines and subsequently challenged with the A20 parental line. After 6 days of in vitro stimulation, lytic activity (as measured by DNA fragmentation) against the 3H-thymidine labeled parental A20 was assessed in a 4 h JAM assay.
Figure 8. Indirect Immunofluorescence profiles of A20 parental and variant lines. A20 parental and variant lines were stained for B7-1, B7-2, and MHC class I and II
molecules.
Solid lines represent staining with isotype controls; stippled lines represent staining with the primary mAb followed by secondary FITC goat anti-rat (B7-1 and B7-2) or streptavidin (MHC I and MHC II) fluorescent conjugate. In this figure, A20/B7-denotes clone A20/B7-2-3. It should be noted that the increase in 87-2 expression in the other A20/B7-2 clones used in various experiments conducted is comparable.
Figures 9. Percent Survival of animals injected with A20 and variant cell lines. 8-10 wks. old female BALB/C mice were subcutaneously inoculated with 105 cells of the parental or variant A20 cell lines on the right flank and survival was subsequently monitored. Kaplan Meier survival statistics were performed as described in Materials and Methods. Events were recorded when animals were euthanized for tumours that had reach 2 cm in diameter or that had ulcerated. Survival for A20/B7-2 clones 3 and 5, A20/mIL- 12, or combinations of A20/B7-2 clones and A20/mIL- 12 are superior and statistically significant (p<0.05) when compared pairwise with the A20 or A20/pRc/pac vector controls.
Figure 10. Percent survival of animals injected with A20/B7-1 and A20/B7-2 variant cell lines. 8-10 wk. old female BALB/c mice were subcutaneously inoculated with 105 cells of the indicated A20 cell variants and survival was monitored. Kaplan Meier survival statistics were performed as described in Materials and Methods.
Events were recorded when animals were euthanized for tumours that had reached 2 cm in diameter or that had ulcerated. Survival for A20/B7-2 clone 3 is superior and statistically significant (p<0.0005) when compared pairwise with the A20/pRc/CMV or A20/B7-1 variant cell lines.
Figure 11. Immunofluorescence staining of tumour cells derived from an animal inoculated with the A20/B7-2 variant. The tumour cells derived from mouse #3669 which had developed and aggressive tumour after inoculation withA20/B7-2-5, the A20 parental cell line, and the A20/B7-2 variant were stained for B7-2 as described in Materials and Methods.
Figures 12. Percent Survival of animals injected with A20 cell variants in a minimal disease model. The A20 parental cells were subcutaneously injected on the left flank. 24 h later the A20 variant cell lines were injected on the opposite flank..
Kaplan Meier survival statistics demonstrated that only the animals inoculated with the clone had a statistically significant increase in survival when compared pairwise with the A20/pRc/CMV (p<0.006) and A20/pRc/pac (p<0.02) vector control groups.
Figure 13. Percent Survival of A20 variant cells in BALB/c nu/nu mice. 105 A20 parental or variant cells were injected subcutaneously and survival was monitored.
Statistically significant differences were not demonstrated when each of the groups were compared pairwise p<0.05.
Figure 14. Generation of CTLs in animals inununized with the A20 variant cell lines.
BALB/c mice were inoculated subcutaneously with 105 A20 parental or variant cell lines.
After 16 days, spleens were harvested and cell suspensions prepared. These lymphocytes were cultured with irradiated A20 cells. After in vitro stimulation, lytic activity (as measured by DNA fragmentation) against the 3H-thymidine labeled parental A20 was assessed in a 4 h JAM assay.
Figure 15. Tumour regression in mice upon termination of CD8+ T cell depletion.
Groups of 5 mice each were depleted of CD8+ T cells 105 A20 tuniour cell variants were subcutaneously inoculated. By day 19, 3 of the mice inoculated with the A20/B7-variant developed progressive tumours. CD8+ T cell depletion was terminated on day 29. By day 37, only one mouse had a tumour while the tumours had regressed in the remaining mice. As previously shown, mice depleted of CD8+ T cells had inoculated with A20B7-1/mIL-12 variants did not develop tumours.
Figure 16. LAK cell lysis of A20 parental and variant lines. LAK cell cultures were prepared from naive BALB/c mice as described in Materials and Methods. 51 Cr labeled cell lines were used as targets. % NK cells as measured by the DX5 market was 42.6%.
CD3 contamination was 0.4%. LAK cell mediated lysis of A20/B7==1 and A20/B7-2 are compared in (A). LAK cell mediated lysis of A20/B7-1 /mIL-12, A20/mIL-12 and A20/B7-2 are compared in (B).
Figure 17. Lysis of A20 targets by NK cells isolated from mice i.v. immunized with the A20 transfectant cell lines. BALB/c nulnu mice were inoculated with A20/pRe/pac, A20/B7-2, A20/B7-1/mIL-12, A20/mIL-12 variant cell lines. Spleens were removed, single cell suspensions were prepared, and NK cells were enriched as described in Materials and Methods. CD3 contamination was < 1% and DX5 staining ranged from 69-77 %. Labeled A20 target cells were than co-cultured with isolated NK cells at varying E:T ratios.
Detatded Description of the Preferred Embodiments Definitions The term "adenovirus" as used herein mean.s any of a group of viruses having the characteristics of an adenovirus. Adenoviruses aze genErally characterized as double-stranded DNA viruses. They have icosahedral capsids with twelve vertices an,d seven surface proteins. The virion is non-enveloped, spherical and about seventy to ninety nm in size. The genome of an adenovirus typically encodes about thirty proteins.
Both strands of adenovzrus DNA encode genes. Transcriptxoxx occurs in three stages -immediate early, early and late.
The tenn "canary pox virus" as used herein means a poxvirus of the genus avipoviras.
Pox viruses can infect many species of birds, and each species of bird may have its own unique species of pox virus (nnynah bird pox virus, canary pox virus, etc_).
The term "fowl pox" as used herein means a poxvirus infection of poultry and other birds characterized by the formation of wart-like nodules on the skixi and diphtheritic necrotic masses (cankers) in the upper digestive and respiratory tracts. The term "fowl pox virus" as used herein means the type species of the genus avipovirus. It is tlie aetiologic agent of fowl, pox.
The term " hematological" as used herein means of or relating to or involved in hematology. "Hematology" as used herein means the brancb of medicine that deals with diseases of the blood and blood forming orgaits.
lIa The term "hemato,poietic" as used herein means pertaining to the formation of blood or blood cells_ The terxza, "intradermally" as used herein means administered into or within the skin.
The term "intranasally" as used herein rneans administered into or within the nose.
The term "intrathecally" as used herein. means administered into or within the fluid-filled spaces between the thin layers of tissue that over the brain and spinal coi-d.
The term "leukaemia" as used herein means a malignant neoplasm of blood-forming tissues charactexized by abnormal proliferation of leukocytes. "Acute leukaemia" as used herein means rapidly progressing leukemia. "Chronic leukaenua" as used herein means slowly progressing leukemia. "Myelocytic leukaemia" as used herein means a malignant neoplasm. of blood-forming tissues marked by a proliferation of xnyelocytes and their presence in the blood. "Lymphocytic leukaemia" as used herein means leukaemia characterized by enlargement of the lyr,rxphoid tissues and Iymphocytic cells in the circulating blood. "Myeloid leukaernia" as used herein means a chronic leukaemia characterized by granular leukocytes. "Lymphoblastic leukaemia" as used herein mean.s a form of lymphocytic leukaemia in which the abnormal cells in the circulating blood are almost totally lymphoblasts (immature white blood cells). "Juvenile myel.o-monocyti.c leukae.rnia" as used herein means a very rare type of childhood leukemia. It is also sometimes called chronic myclogenous leukaemia.
The term "lymphoma" as used herein means a neoplasm of lymph tissue that is usually malignant. There are many types of lymphoma: "Hodgkin's lymphoma" and Nan-hlodgkins lymphomas being two types. The primary difference between Hodgkin's 1lb Lymphoma and Non-Hodgkin's Lymphoma is the presence of a specific abnormal cell, namely, Reed-Stera;berg cells. There are diff.erent names for Hodgkin's disease:
lymphocytic predominance, nodular sel.erosis, mixed cellularity, ly-mphocyte depletion and unclassifxed.
Non-Hodgkin's Lymphoma (NHL) occurs with the malignant (cancerous) growth of B or T cells. Although both types can develop into lymphomas, B-cell lymphomas are much more common than T-cell lymphomas. There are at least 29 different types of NHL each differentiated by the type of cancer cell. Some scientists classify cells by gi:owth rate: indolent refers to slow growth and aggressive refers to fast-growinb cells.
Some also classify NHLs by cell type: T-cel1, B-cell and follicular cell, etc_ "Burkitt's Lymphoma" as used herein means a type of non-Hodgkin.'s lymphoma that most aften occurs in young people between the ages of 12 and 30.
"Anaplastic large cell lymphoma" as used herein means a rare aggressive form of lymphoma that is usually of T cell origin. "Histiocytic lymphoma" as used herein means a malignant tumor of ret-icular tissue.
The term "myeloma" as used herein means a tumor of one or more cells of the bone marrow. Mycloma, is also known as "multiple myelorna" and myelomatosis.
It is a type of cancer arising from plasma cells, which are normally found in the bone marrow.
In rayeloma, one plasma cell becomes defective and multiplies rapidly, to produce too many plasma cells.
The term "orally" as used lrereiuo means administered into or within the rnouth.
The term "polio virus" as used herein means the virus causing poliornyelitis.
"Poliomyelitis" as used herein means an acute viral disease marked by inflammation of nerve cells o the brain stem and spinal cord.
The term "swine pox" as used herein means a variety of the chicken pox, with acuminated vesicles containing a watery fluid. Also referred to as the water pox. Swine pox vinis is a genus of the family Poxv;rirlae, subfamily Chordopoxvirinae, containuing one species, swinepox virus.
The term "tumor burden" as used herein means, the number of cancer cells, the size of a tumor, or the amount of cancer in the body of a mammal or human, "Upregulation" means gene expression levels are increased.
The term "vaccinia rrirus" as used herein means a virus strain used to vaccinate against smaUpox.
Materials and Methods for IL-12 and/or B7-1 Mice. Female BALB/c, C57BL/6, and nu/nu mice 8-10 wk. of age were purchased from Charles River (St. Constant, Que.) and housed at the Animal Facility at the University of Toronto Medical Sciences Building. All experiments were conducted in accordance with the University of Toronto Animal Care guidelines.
Tumour Cells. A20 is a BALB/c B cell lymphoma (Kim et aL, 1979) expressing MHC class 1 and class iI H-2d molecules and was purchased from the ATCC.
Tumour cells were cultured in vitro in RPMI 1640 medium, supplemented with 15% FCS, 5X10,5 lld M 2-ME. Cells were periodically tested for and found to be free of mycoplasma contamination.
DNA-medialed gene transfer. The pRc/CM'V-B7-1 plasxnid was kindly provided by Dr. L. Nadler (Dana-Farber Cancer Institute, Harvard Medical Sehool, MA) and contains the murine 137-1 cDNA in the pRc/CMV expression vector which includes the bacterial neomycin phosphotransferase (neo) gene eonferring resistance to G41$_ A
BioRad'rA
electroporator was used to tran.sfect 20 ug of linearized pRc/CMV-B7-1 cDNA
(digested with Pvu 1) into the A20 B cell lyrnphoma,. The vector coritrol, pRc/CMV, was also linearized with Pvu I and transfected into A20 cells. Selection in G418 at a final coneentration of 500 uglml was initiated 48 h after tcansfeetion. Positive transfectants were further cloned by limiting dilution.
Recombinant IL-12 retrovirus. To achieve simultaneous expression of the 40-kDA
(p40) and. 35-kDa (p35) subunits of mIL-12 as well as a dominant selectable marker from a single retroviral vector, an expression cassette consisting of the mIL-12 p40 and p35 cDNAs (provided by Genetics Institute, Cambridge, MA) (Hawley et al., 1994), separated by a picornavirus inte,rr.xal ribosome entry site (provided by J.
Majors, Washington University, St. Louis, MO) (Ghattas et al., 1991; Hawley et al., 1996) was inserted into the MSCVpac retroviral vector containing the puromycin 1V=acetyl-transferase (pac) gene (Hawley et al., 1994). In the resulting construct, termed MSCVpac-mIL-12, the p40 and p35 cDNAs are under the transcriptional control of the viral long terminal repeat while the pac gene is transcribed from an internal phosphoglycerate kinase promoter. Construction details and the characterization of the MSCVpac-mlL-12 retroviral vector will be reported elsewhere (Lieu et al., 1997)_ Linearized MSCVpac-mlL-12 plasrnid DNA. was electroporated into GP+E-8 6 ecotropic helper-free packaging cells (Markowitz et al., 1988). Viral supernatant was collected 24 h later and used to transduce tunacamycin-treated (0.1 ug/ml for 16 h) GP+E-86 cells (Hawley et al., 1992) and stable transductants were obtained by selection in 1 ug/mL puromycin (ICN Biomedicals, Costa Mesa; CA) for 2 wk. The pooled population of stably-transduced GP+E-86 cells (GP+E-86/MSCVpac-mlL-12) produced recombinant IvISCVpac-mIL-12 at a titer of 8 X 105 puromycin-resistaut C1;U/ni.l when assayed on NIH3T3 fiibroblasts. The corresponding NIH3T3 titer of parentallvlSCVpac virus exported froin a pooled population of stable GP+1r-86 transductants was puromycin-resistant CI'Ulm1. Producer cells were propagated in DME witlt 4.5 g/1 glucose supplemented with 10% calf serum (Life Technoloaies, Gaithersburg, MD) in a humidified atmosphere containing 5% C02 at 370 C.
Viral supernatants for transduction of A20 tumour cells and B7-1 transfectants, were collected from confluent GP+E-86 cultures 24 b after the medium was changed to RPMI
1640 supplemented with 50 uM 2-ME and 15% FCS, centrifuaed at 3000 g for 5 minutes to remove cellular debris, filtered through 0.45 um filters, and used immediately or frozen at -700 C for later use. To generate stable tumour cell transductants constitutively expressing exogenous mIL-12, tun.tour cells growing at a density of 2 X 105 cells/ml were incubated with 6 nzL viral supernatant in the presence of 2 ug/ml polybrene (Sigma, St. Louis, MO). Twenty-four hours later, an additional 6 rnl viral supexnatant containing 2 ug/mi polybrene was added and the incubation continued for a further 24 h.
The znedium was then charnged to fresh RPMI 1640 supplemented with 50 uM 2-ME and 15%
FCS, and 3 ugfrrtl puromycin was added 48 h later.
,lmmunostai-ntng and FACS analysis. Trans.fectants were analyzed with mAb 1.Cx10 (Nabavi et al., 1992) which is a rat IgG2a B7-1- specific mAb purcliased frorn PharMingen (San Diego, CA). A20 parental and variant lines were also stained for GL1 (Freeman et al._, 1993) a rat IgCr2a 87-2 specific mAb, MK-D6 (Watts et al., 1984) an azxti I-Ad r-mAb, 34-1-2S (Ozato et al., 1982) an anti. H-2 TCdDd mAb. These antibodies were purified from culture supematants using protein Cr sepharose=m purchased from Pharmacia (I'iscataway, NJ). After purification, both MK-D6 and 34-1-2S were biotinylated. Cell lines were also stained with FITC-conjugated anti-mouse IgG
(g-chain specific) purchased from Sigma ImmunoChemicals (St Louis, MO). Isotype controls included biotinylated L243 (Lampson et al., 1980) a mouse anti-human Ia, IaG2a antibody, and rat IgG2a isotypic control from CedarLane Laboratories (Hornby, Ontario).
FITC conjugated goat anti-rat IgO purchased froin CedarLane Laboratories (Flornby,Can.) was used for detecting the binding of B7-1 and B7-2.
Bioti.nyated antibodies were detected using FITC conjugated streptavidin purchased frorrt Molecular Probes (Eugene,OR). Stained cells were analyzed on a Becton DickinsonTM flow cytometer.
Mixed Lymphocyte Tumour Czslt rtres. MLR cultures between C57BL/6 lymph node T
cells and the A20 lymphoma variants were prepared as follows. Lymph nodes taken frorn the ingui.nal, axillary and salivary sites were used to raake suspensions of responder cxlls in HBSS supplemented with 5% FCS, and penicillin/streptomycin. Following their elu.tion from a SephadexTM G-10 column, the cells were treated with a cocktail of 1VIAbs wbich included: 3.168 (anti-CD8), 53.6.7 (agti-CD8), RA3-6B2 (anti-B220), (anti-HSA), and Y-3P (anti-A6) all purchased from ATCC. With the exception of 3.168 a11 supernatants obtained from hybridomas rurchased froin the ATCC were purified using protein A or O SepharoseTM ;purchased from Pharmacia (Piscataway, NJ).
After Ab and complement treatment (Low-Tox-MTM, CedarLane, Hornby, Ontario) the viable cells were recovered by buoyant density centrifugation using Lyxnpholyte-Mm obtained from CedarLane ()`Iornby, Ontario). T cell purity as demonstrated by immunofluorescence staining ranged from 90%-95%. In addition, no response was obtained with T
cells plus Con A alone, indicating that APC contamination was minimal (data not shown).
Stimulator A20 variants were irradiated with 10,200 rads and washed once before use.
IV1LR cultur.es of 1X105 T cells and 2X105 A20 variant cells in a total volume of 200 ul RPMI 1640 mediurn supplemented with 10% FCS purchased frorr.x P.A. Biologicals (Sydney, Australia), non-essential amino acids, glutamine, pyruvate, penicill,inlstreptorrtycin, and 2-ME, were prepared in duplicate using 96 well plates (Nunc, cat. 1-67008). The cultures were incubated for 3 d at 370C and 5% C02 in humidified conditions, at which time 50 ul of inediLun was removed and replaced with 50 ul of fresh medium. 48 h later, the contents of duplicate cultures were pooled, centrifuged, and 300 ul of supernatant was collected and frozen at -70oC.
Bioassays. Bioassays for both IL-2 and IL-4 were performed identically with the exception that IL-2 was detected using the CTLL cell line and IL-4 was detected using the CT.4S cell line (Hu-Li, et al., 1989). The CT.4S cell line was kindly provided by Dr.
W. Paul (National Institutes of Health, Bethesda, MD). Anti-IL-4 mAb 11B11 purchased from the ATCC, used at 4 ug/ml was added to all cultures containing CTLL
cells. Both the CTLL and CT.4S lines were maintained in continuous culture using rIL-2 supernatant or rIL-4 supernatant, respectively, derived from the lymphokine-secreting X63 cell line series, as developed by Kanagawa and Melchers (Kanagawa and Melchers, 1988).
Two-fold dilutions of supematant, starting at 1/4 were prepared in triplicate and indicator cells were cultured for 24 h in 100 ul using 96 well plates. During the last 8 h, the cells were labeled with 3H-thymidine (Amersham, 5 Ci/mmol), added at I uCi per well. Cultures were processed for liquid scintillation counting and the amount of incorporation was determined using the Packard Top Count system. For inhibition of cytokine production, anti-B7-1 mAb 1610A1 kindly provided by Hans Reiser (Dana-Farber Cancer Institute, Harvard Medical School, MA), anti-B7-2 GL 1(ATCC), or control rat Ig purchased from Sigma (St.Louis, MO.) or control hamster Ig purchased from Jackson (West Grove, PA) were added to the cultures.
ELISAs. Mouse IL-12 was quantified by using the ELISA mouse IL-12 Cytoscreen kit (Biosource, Camarillo, California) and performed as described by the manufacturer's instructions. 1 X 106 of variant cells were plated in complete media and supernatants that were used for the ELISA were collected 24 h later. Supernatant levels of IFN-y were measured by capture ELISA using anti-murine IFN-y MAbs purchased from PharMingen (San Diego, CA). ELISA was performed as described by the manufacturer's instructions, using the diluted supematant.
Animal Studies. For tumourigenicity experiments, A20 parental and variant lines in log phase were harvested, washed, and resuspended in 0.2m1 RPMI 1640 medium lacking any supplements. 105 cells were injected subcutaneously in the right flank.
For challenge experiments, animals found free of tumour were either boosted and then challenged 10 days after the boost or challenged without boosting with 105 A20-parental cells injected subcutaneously on the left flank. In minimal disease inodel mixing experiments, 105 A20 parental and 105 A20 variant cells were mixed and injected subcutaneously at the same site (right flank) or at different sites (right and left flank). In all animal studies, 10 ear-tagged animals were injected for each variant cell line. 5 animals each were injected to assess the tumourigenicity of the A20/pRc/pac and A20/B7-1/pac vector controls. Tumour incidence and size were monitored 3 times a week. Animals were euthanized when the tumour diameter was approximately 2 cm or tumour ulceration was apparent.
For injections into nu/nu mice, 105 A20/pRc/pac vector control, A20/mIL-12, and A20/B7-1/mIL-12 variants were subcutaneously inoculated. Five animals were used for each variant group.
Jam Test. The Jam CTL test was performed as described by Matzinger (Matzinger, 1991). Briefly, single cell suspensions were made from spleens derived from mice that had been previously immunized with the IL- 12 and B7-1 /IL-12 variants and had furthermore rejected challenge by the parental A20 unmodified line. These responder cells were incubated with irradiated A20 parental cells for 6 days. Viable effector cells were then incubated with 3H-thymidine (Amersham) labeled A20 target cells at different E:T ratios in a 96 well plate (Nunc, cat. 1-63320) for 4 h at 37 oC. After 4 h, the cells were harvested with a cell harvester and counted by scintillation counting. %
specific killing was calculated as follows: (Spontaneous cpm-Experimental cpm)/(Spontaneous cpm) X 100. Cpm were taken of target cells alone both at to and t4 to assure that target cells were not undergoing spontaneous cell death. The standard error of the mean did not exceed 5.5 %. As controls, C57BL/6 mouse spleens were used as responders and BALB/c mouse spleens were used as stimulators in an allogeneic mixed lymphocyte reaction. Naive BALB/c mouse spleens incubated with the parental A20 cell line was used as a negative control. In addition, animals were immunized with the A20/pRc/pac vector control and two weeks later the spleens were dissected and used for JAM
assays.
Animals immunized with the A20/pRc/pac vector control at the same time as the variants could not be assessed because these vector control animals had to be euthanized within three weeks of the experiment reaching tumour diameters of 2 cm.
In-vivo Depletion studies. Ascites fluid of hybridoma 2.43 (anti-CD8) (ATCC;
TIB
210), and GK1.5 hybridoma, L3T4) (ATCC; TIB 207) were kindly provided by Dr.
K.
Irvine (National Cancer Institute, Bethesda, MD). BALB/c mice were i.v.
injected with empirically detennined levels of 2.43 and GK1.5 diluted in 0.5 mL of PBS four days prior to tumour inoculation and then weekly i.p. thereafter (Rao et al., 1996). Inguinal, axillary and salivary lymph nodes were dissected from control animals to assure >90%
depletion of T cell subsets using indirect immunofluorescence staining with FITC
conjugated mAb CD4, CD8 antibodies purchased from Pharmingen (San Diego, CA), or goat anti-rat FITC purchased from CedarLane (Hornby, Can.). The tumour cell variants were inoculated four days after the initial antibody injection.
Statistical Analysis. Kaplan Meier survival curves were compared using the log rank test in the SPSS 6.1 Macintosh Version statistical package. Differences are considered statistically significant when p<0.05.
Results for IL-12 and/or B7-1 Generation of A20 variants. The A20 murine B-cell lymphoma is IgM-, IgG+, IgA-, Ia+, Fc receptor+, and complement receptor- (Kim et al., 1979). A20 also expresses both MHC class I and class II molecules, and low levels of B7-2 as determined by indirect immunofluorescence staining (Fig.l). B7-1 expression, however, is undetectable. A20 variants expressing both low and high levels of B7-1 were generated by transfection with the murine B7-1 cDNA and selection in G418. Vector control variants were generated by transfection with pRc/CMV. Positive independent clones were obtained by limiting dilution. Because of the reported in vitro enhancement of B7-1 costimulation of T cells by IL-12 (Gajewski et al., 1995), we also introduced mIL-12 using the MSCVpac retroviral vector system (Hawley et a1.,1994). Preliminary studies had shown no significant anti-tumour effects using the A20/B7-1-low expressing variant, hence, the populations used secreted mIL-12 at levels of 1, 250 pg 7.81 pg /ml/106 cells/24 h.
Immunostaining and FACS analyses demonstrated that expression of the costimulatory molecules, B7-1 and B7-2, as well as MHC class I and class II, were stable in all variant lines (Fig. 1). The phenotypic profiles of these variants did not charige upon reassessment after several weeks in culture. In addition, the doubling rates of the A20-parental and variant lines were not significantly different (data not shown).
Costimulation and Cytokine Production ofAllogeneic CD4+ TLymphocytes by A20/B7-1 and A20/B7-1/mIL-12. T'o assess the costimulatory function of the A20 variants, A20 parental and variant lines (H-2d) were compared in their ability to activate purified allogeneic CD4+ T cells in a mixed lymphocyte tumour culture assay, and IL-2, IL-4, and IFN-y secretion by the reacting T cells was assessed. Only the (high) and A20B7-1 /mIL-12 variants were able to stimulate allogeneic (H-2b) CD4+ T
cells (Fig. 2 A, B) to secrete IL-2 and IL-4. IL-2 or IL-4 secretion by allogeneic T cells was not seen by the A20/B7-1 (low), A20/mIL-12, or parental tumour lines despite the low endogenous level of B7-2. Interestingly, both the levels of IL-2 and IL-4 secretion by T cells was reduced in the A20/B7-1 /mIL-12 mixed lymphocyte tumour culture as compared to the culture containing the A20/B7-1 variant.
IFN-y secretion was also assessed after stimulation of allogeneic CD4+ T cells with the A20 parental and variant lines. Only A20/mIL-12 or A20B7-11mIL-12 variants were able to induce allogeneic T cells to secrete IFN-y (Fig. 2 C). Both variants induced the T
cells to secrete comparable levels of IFN-y.
IL-2 secretion was completely inhibited when anti-B7-1 was added to the mixed lymphocyte tumour cultures consisting of purified allogeneic CD4+ T cells and 1 or A20/B7-1/mIL-12 variant lines (Fig. 3). Anti-B7-2 had no or minimal effects on reducing IL-2 secretion when added to either the A20/B7-1 or the A20/B7-1/mIL-cultures respectively. Similar elimination of IL-4 secretion was seen when antibodies to 137-1 were added to the mixed lymphocyte tumour cultures (data not shown).
A20 Transfectants Expressing B 7-1 Delay Tumour Onset and Prolong Survival While A20/mIL-12 or A20/B7-1/mIL-12 are Rejected by BALB/c Mice. 105 cells of each variant line were injected subcutaneously into the right flank of BALB/c mice. While animals receiving A20B7-1 cells develop tumours, the tumour onset is delayed. That is, 80% of animals receiving control A20/pRc/CMV cells have palpable tumours by day 18 post injection, while only 40% of animals receiving the A20/B7-1 transfectants have palpable tumours at this time point (data not shown). In addition, B7-1 prolongs survival (Fig.4).
While 50% of the animals receiving the A20/pRc/CMV vector control variant were euthanized by day 27, 50% of the A20/B7-1 recipients were euthanized by day 37.
Strikingly, the animals receiving the A20/mIL-12 or A20/B7-1 /mIL-12 variants do not develop tumours (Fig. 4). These results suggest that although B7-1 reduces the tumourigenicity of the A20 line, expression of IL- 12 or co-expression of B7-1 results in complete rejection.
Inoculation of 1'umour cells Expressing mIL-12 With or Without B7-1 Protects Against Subsequent Challenge With Unmodifted Parental Tumour Cells. Animals from the above experiment that received 105 A20/B7-1 /mIL- 12 or A20/mIL- 12 did not develop tumours. 45 days post initial injection, 50% of the tumour free animals were boosted with either the A20/87-1/mIL-12 or the A20/m1L12 variant respectively at the same site of initial subcutaneous injection. Ten days following the boost, all tumour free animals boosted and non-boosted were challenged subcutaneously on the alternate left ilank with 105 parental A20 nontransfected cell line. Tumour-challenged mice were observed for tumour incidence and survival. At this dose, both A20./B7-1/mIL12 and A20/mIL- 12 were equivalently able to protect mice against subsequent tumour challenge.
By day 41, post tumour challenge, one mouse boosted with the A20/mIL- 12 variant developed a tumour, while two mice boosted with the A20/B7-1/mIL-12 variant developed tumours. Also, one non-boosted mouse from the A20/B7-1/mIL-12 group developed a tumour. It should be noted that these tumours developed on the site of challenge with the parental cell line, i.e., the left flank. Overall, 70% and 90% of the immunized mice from the A20/B7-1 /mIL-12 and A20/mIL- 12 groups respectively, remained tumour free after having been re-challenged with a lethal dose (105 cells) of the unmodified parental line (data not shown).
A20/B7-1/mIL-12 Variants Result in Prolonged Survival In a Minimal Disease Model. In this series of experiments analogous to the clinical setting of minimal disease, mice were immunized with genetically modified tumour variants on the same day as the parental tumour cells were inoculated. Immunizations were performed at the same site (intratumourally) or at distant sites. Figure 5 shows the results of one such experiment where 105 A20 parental cells were injected into the left flank, and 105 cells of a particular A20 variant line were injected into the right flank. Figure 5 illustrates that by day 46 only one animal remained in the pRc/CMV vector control group, while no animals receiving the pRc/pac cells were alive. Also, protection was not observed in animals that were immunized with A20/B7-1 on the opposite flank. Tumours developed in both flanks in all of these animals. In contrast, both the A20/mIL- 12 and I/mIL-12 variants delayed tumour growth of parental A20 cells at the remote site.
However, only the A20/87-1 /mIL- 12 vaccination induced statistically significant increases in survival when compared to the vector control treatment groups (p<.02).
Resultant tumours in these treatment groups grew only in the flank receiving the A20 parental cells.
The above experiment was modified by inoculating a mixture of the variant tumour cells used for immunization with the parental tumour cells so as to resemble the clinical situation of intratumoural vaccination into a site of minimal disease. 105 A20-parental and 105 A20 variant lines which included A20/pRc/CMV, A20/pRc/pac, A20/B7-1, A20/B7-1/pac, A20/87-1/mIL-12, and A20/mIL-12 were mixed and injected subcutaneously into the right flank and tumour incidence and survival were monitored.
Progressive tumours grew in all animals receiving vector transduced control or the B7-1 cells and A20 parental cells at the same site. Under these conditions, the variant did not produce a delay in tumour onset (data not shown). Similarly, by day 25, two out of ten animals receiving the A20/pRc/mIL- 12 variant had palpable tumours and by day 42, 80% of these animals had progressive tumours. At the day 42 time point, 100% of the animals inoculated with the A20/B7-1/mIL-12 variant were tumour free. By day 66, only 2 tumour free mice remained in the A20/mIL- 12 treatr.nent group while 80%
of the animals receiving the A20/B7-1 /mIL-12 variant were alive and 6 out of these 8 animals were tumour free (Fig. 6). These results suggest that at these doses, the A20/B7-I/mIL-12 variant was most effective in preventing outgrowth of unmodified parental tumour cells when introduced into the vicinity of the parental tumour cells at the same site of injection.
Mechanisms Mediating Anti-tumour Immunity. Animals that had been initially immunized with 105 A20/B7-1/mIL-12 or 105 A20/mIL-12 variant lines subsequently rejected challenge with 105 parental unmodified A20 cells on the opposite site. To gain insight into the mechanism responsible for this observed in vivo anti-tumour activity, cytolytic T cell activity was assessed in these animals and compared to that in naive animals or animals immunized with the A20/pRc/pac vector control. Results demonstrated that animals immunized with the A20/mIL-12 variant had a specific lysis of A20 targets of 76.8%, while animals immunized with the A20/B7- 1/mIL- 12 variant had a lysis of 72.8% both at an E:T of 40:1 (Fig. 7). At this ratio, animals immunized with the A20/pRc/pac variant or naive mice demonstrated less than 50% of this lytic activity against the A20 parental line.
To gain insight into the particular T' cell subsets involved in mediating anti-tumour immunity, BALB/c mice were depleted of CD4+ or CD8+ T cells and inoculated with the A20/pRc/pac, A20/mIL-12, and A20/B7-1/mIL-12 variant tumour lines. By day 27, tumours had developed in untreated animals or animals depleted of CD4+ or CD8+
T
cells inoculated with A20/pRc/pac tumour cells (Table 1). Only 2/5 animals depleted of CD4+ T cells and inoculated with the vector control, A20/pRc/pac, developed tumours.
It should be noted, however, that statistically significant differences in survival, are not achieved in the untreated or depleted animals that have been inoculated with the vector control. All animals receiving the IL-12 or B7-1 and IL-12 variants, however, did not develop tumours suggesting that the enhanced immunogenicity induced by the IL-variants could be mediated by both CD4+ or CD8+ T cells or by cells other than T
lymphocytes. Another tumourigenicity experiment was performed using mice deficient in both CD4 and CD8 T cells, nu/nu mice. 105 A20/pRcpac vector control, A20/B7-1, A20/mIL-12, A20/B7-1/mIL-12 variant lines were injected subcutaneously into these mice and tumour development was monitored. In this experiment enhanced immunogenicity was not seen in mice inoculated with the IL- 12 variants (data not shown). Taken together, these data suggest that either CD4+ or CD8+ T cells may mediate the enhanced immunogenicity elicited by the IL-12 variants and that this activity is lost in mice deficient in both CD4+ and CD8+ T cells.
I)iscussion for IL-12 and/or B7-1 Approaches to enhance the immunogenicity and reduce the tumourigenicity of tumours are actively being tested in various murine tumour models. These approaches have included gene transfer of costimulatory molecules such as B7-1 or various cytokines ('Tepper et al., 1989; Fearon et al., 1990; Dranoff et al., 1993; Pardoll, 1995). Several cytokines have been able to reduce the tumourigenicity of the tumour cell and even confer protection against unrnodified parental tumour cells (for review see Colombo et al., 1994). Similarly, reduction in turxmourigenicity attd protection against uni-oodified parental tumour cells has been observed through the introduction of B7-1 (Chen et al., 1992; Baskar et al., 1995; Townsend et al., 1993)_ A strategy to enhance the imxnunobenicity of the A20 murine B cell lympholna has been studied by introducing both the genes for B7-1 and IL-12 into the same tumour cells. The A20 B ce111ytnphoma murine tumour model is a good model to study such a strategy in, that it expresses IgG, N,QIC I and ir molccules ajid levels of ezzdogenous 137-2, yet, it is tumourigenic. In fact, subcutaneous inoculation of 105 tumour cells proliferate and kill rnice within 25 days.
Purified, allogeneic CD4+ T cells secreted IL-2 and IL-4 in the presence of A20 cells altered to express high levels of B7-1. The secretion of these cytokines in the presence of B7-1 is consistent with work from previous groups (Galvin et al_,1992). The specifieity of these increases was shown by elimination of this cytokine secretion when antibodies to B7-1 were added to the cultures. Interestingly, despite the low levcls of B7-2 expression by the parental A20 cells, this level of expression was not adequate to costimulate T cells to secrete IL-2 or II,-4. In addition, antibodies to B7-2 did not significantly reduce the IL-2 or XL.-4 secretion induced by the A20/B7-1 or A20B7-1/m1L-12 variants.
Because B7-2 has been shown to costimulate T cells (Chen, C. et aI.,1994), a threshold level of 87-2 expression may be necessary for this response and that A20 cells express sub-threshold levels of this molecule.
IL-12 variants either with or without B7-1 induced marked increases in XpN-y secretion by allogeneic T cells. The levels of IF'N-y induced were comparable and not statistically significantly different between the A20/mIL-12 or the A20/B7-1/mIL-12 variants indicating that the different in vivo effects between the A20/rnlL-12 or A20/B7-I/mIL-12 variants is not likely due to differences in the in vxvo levels of IL-12 secretion between these independently derived IL- 12 lines. The amount of both IL-2 and secreted by allogeneic T cells was reduced in the cultures consisting of the ,,4,20/B7-1/mIL-12 variarnts. Others have shown that IL-12 plays a role in cell-mediated immunity via the regulation of Thl and Th2 subsets (Romagnani et al., 1992; Germann et al., 1993;
Manetti et al., 1993). Moreover, IL-12 promotes the production of IFN-y both in vitro (Chan et al., 1991) and in vivo (Brunda et al., 1993). Because IL-12 has been shown to promote the generation of Th1 cells (Cardinb et al., 1989), it was not unexpected to see reduced IL-4 secretion in the presence of IL-12. The explanation for the reduction in IL-2 secretion, however, is unclear.
Indeed, the tumourigenicity of the A20 cells was reduced and mice immunized with the A20/B7-1 variant had delayed ttunour onset. These results are consistent with results expected for modification of relatively non-immunogenic tumour cells with the costimulatory molecule (Chen, L. et al., 1994). My preliminary studies have shown that animals irnraunized with variants that express only low levels of I37-1 develop progressive tumours at a similar rate to the parental line whereas the high expressing cell line significantly delays tumour onset (data nat shown).
By direct comparison, tturnourigenicity was strikingly reduced when IL-12 was introduced into the A20 vector control and A20/B7-1 high expressing variants.
Syngeneic animals completely rejected the A20/rnIL-12 or A20/B7-1/mIL-12 modified tumour cells. 80% of these animals were protected against subsequent challenge with the parental tumour line. In addition, these data demonstrate that activation of CTLs may play a role in the marked reduction in the tumourigenicity of the A20/B7-1/mIL-12 or A20/mIL- 12 variants.
In a more stringent experiment analogous to the minimal disease state, the A20 parental cells and the A20 variant lines were injected at the same time and at the same site. Progressive tumours grew in all animals, except in those animals receiving the A20B7-1 /mIL- 12 variant.
In addition, while all other treatment groups, including the A20/B7-1 group developed tumours by day 16 post injection, tumour onset was delayed in the animals receiving the A20/mIL- 12 variant at the same site. However, most animals receiving the A20/mIL-12 tumour cells eventually developed progressive tumours and by day 66 post injection only 20% survived. At this same time point, 8 out of 10 animals inoculated with the A20/B7-1/mIL-12 variant and the parental cells at the same site were alive, and 6 of these animals were still tumour free.
In the most stringent experiment, the A20 parental, unmodified tumour cells were inoculated at the same time but at a different site than the A20 variant lines. Survival of the group that received the A20/B7-1/mIL-12 variant was superior, although not statistically greater than the animals receiving the A20/mIL-12 variant. The therapeutic advantage of these variants may be greatest in a minimal residual disease setting when variant tumour cell immunogens are inoculated into sites of residual disease.
The mechanism of the enhanced anti-tumour activity was complex and probably multifactorial. Increased CTL activity was detected in mice that survived tumour challenge with the IL- 12 or B7-1 and IL- 12 variants compared to un-immunized mice or mice immunized with vector control cells. Tumourigenicity experiments performed in nu/nu mice showed that the enhanced anti-tumour activity was lost in these mice. On the other hand, the T cell depletion experiments showed that the enhanced anti-tumour activity seen in IL- 12 or B7-1/IL-12 variants was not lost when either CD4+
or CD8+
lymphocytes were depleted from BALB/c mice. This suggested that either T cell subset could mediate this anti-tumour activity or that some non T cell such as NK
cells were participating. Recently, studies suggest that B7-1 may enhance the anti-tumour activity of NK cells (Wu et al., 1995; Chambers et al., 1996) and IL-12 is well known to increase NK activity (Kobayashi et al., 1989; Robertson et al., 1992; Naume et al, 1992; Gately et al., 1991, 1992). The in vitro experiments also showed that there was a significant increase in the cytokines IL-2, IL-4 and IFN-y secretion from CD4+ T cells responding to the IL-12 or B7-1 /IL-12 variants. Taken together these results suggest that the enhanced anti-tumour activity generated by immunization with tumour cells modified with with or without B7-1 is due to the multiple and pleiotrophic effects induced by these genetically modified tumour cells upon the immune system.
Potent anti-tumour effects of recombinant IL- 12 or localized paracrine IL- 12 expression have been reported previously (Brunda et al., 1993; Tahara et al., 1995;
Tannenbaum et al, 1996; Nastala et al., 1994; Zitvogel et al., 1995). However, the use of tumour cells genetically modified to express both B7-1 and IL-12 at low levels would appear to offer distinct advantages over the former strategy, i.e., the potential for aximizing tumour immunogenicity (Nanda et al., 1995) while minimizing any possible side-effects associated with high circulating cytokine levels (Cohen, 1995).
These data demonstrate that tumour cells modiFied with IL-12 were superior to tumour cells modified with B7-1 alone in conferring protection. Both B7-1 and IL-12 may allow for the recruitment and activation of both Thl. and Th2 lyn-lphocyte populations and subsequent alterations in the local cy-tokine profile causing the generation of cytolytic T
lymphocyte activity. In addition, these studies provide the first formal description of anti-tumour activity with tumour cells modified to express both B7-1 and secrete IL-12.
Manipulation of the cooperative mechanisms which may be triggered by the cornbination of costimulatory and cytokine activity may be a novel and effective therapeutic approach in patients with cancer. The methodology of same site versus different site illustrates the potential of these genetically modified tumour cells as anti-tumour vaccines.
Materlals and Methods for B7-2 Mice_ Same as for IL12 and/or B7-1.
Tumour Cell s_ Same as for IL 12 and/or B7-1.
Gene Transfer. The pRc/C1VIV-B7-1 plasmid was kindly provided by Dr. L. Nadler (Dana Farber Cancer Institute, Harvard Medical School, MA) and A20/B7-1, A20/pRc/CMV variants were generated by electroporation as previously described (Pizzoferrato et al., 1997). The mouse B7-2 cDNA was cloned from cellular RNA
extracted frorn, db-cAMP stimulated M12 cells (Kim et aI., 1979) by RT-PCR
using prinaers derived from the published sequence (Freeman et al., 1993): 5'-ctgtagaegtgttecagaacttaeggaa-3' and 5'- caatttggggttcaagttectteagg-3' and internal primer 5'-taaattagtta.tca.gaaa-3'. The arnplified product was subcloned into the pRc/CMV
expression vector (Xba I and Not I sites ) and sequenced to rule out PCR
errors. To ensure for appropriate protein expression, COS cells were transiently transfected using lipofectanrzineT~4 (GibcoBRL, Life Technologies, Gaithersbuarg, MD) and transfection was performed as described by the nctanufacturer's instructions. B7-2 expression was assessed by irzzznunofluorescence 72 h post transfection. The linearized pRc/CMV-B7-2 cDNA
(20 g or 30 g digested with Pvu I) was then electroporated into A20 cells in independent transfections using a Bi.oRadT"' electroporator. These cells were selected in G418 at a final concentration of 500 g/mL 48 h after transfection. Positive transfectants from bulk cultures were further cloned by limiting dilution. Generation of the A20/mIL-12 and A20/B7-1/mIL-12 variants has previously been described (Pizzoferrato et al., 1997).
fnxmunostaining ancl FACS analysis. Same as for IL12 and/or B7-1.
Animal Studies. For the first tumourigenicity study, 105 live A20/pRc/pac vector control, A20/$7-2 clones 3 and 5, A20/rraIL-12, or a combination of A20/rnIL-12 and A20/B7-2 variants in log phase were harvested, washed, and resuspended in 0.2 mL
RPMI 1640 medium lacking any supplements. Animals were injected subcutaneously into the right flank and monitored for tumour incidence and survival. Sixty-five days post injection, surviviria animals were challenged on the opposite flank with 105 live A20 parental cells. In the second turn,ozigenicity experiment, 105 A20/pRc/CMV
vector control, A20B7-2 clones X and 5 and cells derived from A20/$7-2 bulk cultures were inoculated into the right flank of BALB/c mice. Fifty-six days post immunization, surviving animals were challenged with the parental A20 cells on the opposite flank.
Naive aninSats injected with the parental line were used as controls. In the third turnourigenicity experiment, BALB/c rni.ce were inoculated with 105 A20/pRc/CW
vector control cells, A20/B7-2 clone 3, or A20/B7- I variants and monitored for survival.
For established disease experiments, 105 A20 cells were inoculated into the left #lank of BALB/c mice and 24 h later, 105 cells of each variant were injected into the right ,Oank.
For all the above experiments, 10 ear-tagged animals were used for each experimental group. For injection into nude animals, 105 A20/B7-1, A20/B7-2 clone 3, or A20/pRc/CMV were inoculated into BALB/c nu/nu mice and survival was monitored.
Five animals were used for each group. All animals were euthanized when tumours reached 2 cm in diameter or began to ulcerate.
Jam test. The Jam CTL test was performed as described by Matzinger (Matzinger et al., 1991). Briefly, spleens were dissected and single cell suspensions were made from BALB/c mice immunized with 105 A20/pRc/pac vector control, A20/B7-2 clones 3 and 5, A20/mIL-12, or a combination of 105 A20/B7-2 clones 3 or 5 and 105 A20/mIL-variants on the right flank. These responder cells were incubated with irradiated A20 parental cells. Viable effector cells were then incubated with 3H-thymidine (Amersham) labeled A20 target cells at different E:T ratios in a 96 well plate (Nunc, cat.l-63320) for 4 h at 37 C. After 4 h, the cells were harvested with a cell harvester and counted by scintillation counting. % specific killing was calculated as follows:
(Spontaneous cpm -Experimental cpm)/(Spontaneous cpm) x 100. The standard deviation of the mean did not exceed 10 %.
As controls, C57BL/6 mouse spleens were used as responders and irradiated BALB/c mouse spleens were used as stimulators in an allogeneic mixed lymphocyte reaction.
Naive BALB/c mouse spleens and spleens derived from mice immunized with the vector control variant incubated with the irradiated parental A20 cell line were used as negative controls. Cpm were taken of target cells alone at to and t4 to confirm that spontaneous cell death was not occurring. Also, other studies were conducted in which viable effector cells incubated at different E:T ratios with labeled targets were harvested at to to confirm that the labeled target cells were not spontaneously dying.
Depletion of T cell Subsets. Same as for IL 12 and/or B7-1.
Preparation of NK effectors from immunized animals: Spleens were pressed through a wire mesh screen with a disposable syringe plunger into complete medium (CM) which is a-MEM (Gibco, BRL, Burlington, Ontario, Canada) containing 10% FCS (Gibco, BRL), 50 M 2-ME and 10 mM HEPES. Released cells were layered over 5 ml of 6%
BSA in PBS and centrifuged to remove cell debris. The splenocytes were loaded onto a nylon wool column (Julius et al., 1973) and incubated for 1 hr at 37 C. NK
cells were enriched (69-77%) in the nylon wool non-adherent fraction, and were used as effectors in a 51Cr-release cytotoxicity assay. Anti-CD3-phycoerythrin conjugated (clone 29B) purchased from Sigma (St.Louis, MO) and DX5-FITC mAb which is a novel pan-NK
cell marker that binds an as yet unknown surface molecule on NK cells from mice of all strains tested to date, purchased from Pharmingen (San Diego, CA) were used to assess the purity of the isolated NK population.
Preparation of lymphokine activated killer culture (LAK): splenocytes from BALB/c nulnu mice were suspended in 10 ml CM, supplemented with 500 U/ml murine rIL-2 obtained from a cell line transfected with the mouse IL-2 gene (Karasuyama et al., 1989). The cells were incubated at 37 C, 10% C02 /air atmosphere for 2 days.
On the day of experiment, both adherent cells and nonadherent cells were collected, washed and resuspended in 5 ml CM, underlaid with 5 ml lympholyte-M (Cedarlane Laboratories, Hornby, Ontario), and centrifuged at 500 g for 20 min to remove dead cells.
The viable cells were washed, resuspended in CM, and used as effectors in 51 Cr-release cytotoxicity assay.
Preparation of NK targets: YAC-1 (murine lymphoma, TIB 160) and P815 (murine mastocytoma, TIB64) were obtained from the ATCC. They were grown in CM and passed three times a week. A20 and A20 transfectants were prepared as described previously.
51Cr release cytotoxicity assay: Target cells (1 x 107) were labeled with 0.4 mCi of sodium 51 Cr-chromate (Dupont Chemicals, Mississauga, Ontario, Canada) for 1 hr.
After three washes in CM, the target cells were added to 96-well V-bottomed microtiter plates in 100 l aliquots. LAK effectors (in 100 l ) were added to the radiolabeled targets at various E/T ratios in replicates. The plates were centrifuged at 400 rpm for 5 min. After a 4 hr-incubation at 37 C, the plates were centrifuged at 700 rpm and the supernatants were harvested for determination of radioactivity levels in a gamma counter.
Specific lysis was calculated as described elsewhere (Kung and Miller, 1997).
Statistical Analysis. Kaplan Meier survival curves were compared using the log rank test in the SPSS 6.1 Macintosh Version statistical package. Differences were considered statistically significant when p<0.05.
Results for B7-2 Generation of A20 variants. The A20 murine B-cell lymphoma is IgM-, IgG+, IgA-, Ia+, Fc receptor+, and complement receptor- (Kim et al., 1979). A20 also expresses both MHC class I and class II molecules, and intermediate levels of B7-2 as determined by indirect immunofluorescence staining (Fig.8). B7-1 protein expression is undetectable.
Variants expressing B7-1, B7-1/mIL-12, and mIL- 12 were described previously (Pizzoferrato et al., 1997). The B7-1 and B7-1/mIL-12 variants had comparable levels of 137-1 protein expression as measured by immunofluorescence staining (Figure 1). Vector control variants were established by transfection of pRc/CMV and retroviral infection of MSCV/pac (Pizzoferrato et al., 1997). Variants expressing B7-2 at levels higher than that seen in the parental A20 cell line were generated by transfection with the pRc/CMV/B7-2 plasmid. All positive clones were selected in G418 and further subcloned by limiting dilution.
Preliminary allogeneic mixed lymphocyte reactions were conducted using purified CD4+ T cells from lymph nodes derived from C57BL/6 mice. These T cells were stimulated with the A20-parental, A20/pRc/CMV, and A20/B7-2 bulk clone. IL-4 secretion was assayed using the CT.4S cell line and has previously been described (Pizzoferrato et al., 1997). Both the A20-parental and A20/pRc/CMV lines stimulated CD4+ T cells to secrete IL-4 resulting in CT.4S proliferation. The A20/B7-2 variant expressing more than two fold the level of B7-2 resulted in a statistically significant increase in CT.4S proliferation (data not shown). These data suggest that the endogenous B7-2 expressed by the A20 parental cell line is functional and that even slight increases in B7-2 expression could enhance T cell activation in vitro.
The clones selected for the in vivo studies (A20/B7-2-1, A20/B7-2-3, A20/B7-2-5) expressed approximately 2.5X more B7-2 protein expression than the parental A20 clone as indicated by immunofluorescence staining. Immunofluorescence staining indicated that expression of surface molecules MHC class I and II was unaltered in these clones (Fig.8). The phenotypic profile of these variants did not change upon reassessment after several weeks in culture. Moreover, the doubling rates of the A20-parental and variant lines were not significantly different (data not shown).
A20 Transfectants Expressing B7-2, and/or m1L-12 are Rejected By Syngeneic BALB/c mice and induce protective systemic anti-tumour immunity.
105 live tumour cell variants including A20B7-2-3, A20/B7-2-5, A.20/mIL-12, or a combination of A20/B7-2 transfectants clone 3 or 5, and A20/mIL- 12 transfectants were injected subcutaneously into 10 mice each. Animals inoculated with the vector control or parental A201ine developed progressive tumours and most of these mice were euthanized by day thirty-six (Fig.9). The A20/mIL- 12 transfectant was completely rejected by BALB/c mice. Of the animals inoculated with either of the two A20/B7-2 clones, only two animals developed a tumour and this difference is statistically insignificant when compared to the survival curves of the IL-12 variant (p>0.05) (Fig.9).
Coinjection of the A20/B7-2 and A20/mIL-12 variants did not result in a statistically significant improval in survival when compared to survival of mice injected with either one of these variant lines. 65 days post the initial inoculation the surviving animals were challenged with 105 A20 parental cells on the opposite flank. By day forty-five, 6 out of the 9 previously unimmunized control animals had developed tumours and were euthanized. One animal from the group initially immunized with the A20/B7-2 clone 5 group developed a tumour and was euthanized on day thirty-five. All other animals inoculated with the transfectants and challenged with the unmodified parental cell line did not develop tumours (data not shown).
In a repeat of this experiment, mice were injected with 105 cells from either two independent A20/B7-2 bulk cultures, A20/B7-2 clone 1 or 5, or A20/pRc/CMV
vector control variants. Only animals inoculated with the A20/pRc/CMV vector transfectant developed progressive tumours. Fifty-six days post initial inoculation protected animals were challenged with parental cells on the opposite side. By day thirty-five post challenge, only one mouse initially immunized with the A20/B7-2-5 variant clone had developed a tumour. In contrast, only 20% of the animals in the control nonimmunized group were still alive by day forty-three (data not shown). Tumours did not develop in any of the other animals (data not shown).
In both of the above challenge experiments, mice inoculated with the A20/B7-2 variants had statistically significant increases in survival over animals immunized with the parental or vector control A20 cell lines. Also, there was no difference in survival when the animals were inoculated with the A20/B7-2 variants alone or with a combination of A20/B7-2 and the A20/mIL-12 variants. Collectively, these tumourigenicity studies suggest that tumour cells genetically modified with B7-2 induce anti-tumour responses and that these are as potent in inducing anti-tumour immunity as tumour cells genetically modified with IL-12.
B7-2 is more effective than B7-1 in the A20 B cell lymphoma murine model system.
To compare the effectiveness of A20/B7-1 and A20/B7-2 in a vaccination animal tumour model, 105 A20/B7-1, A20/B7-2 clone 3, and A20/pRc/CMV variants were inoculated into 10 animals each. The animals inoculated with the B7-I or vector control variant developed progressive tumours and 90 % were euthanized by day thirty-eight. In contrast, all animals inoculated with the B7-2 clone remained tumour free with a statistically significant increase in survival when compared pairwise with the animals injected with the A20/pRc/CMV vector control (p<0.0005) or animals inoculated with the A20/B7-1 variant (p<0.0005) (Fig. 10). Statistically significant differences in survival were not observed between animals injected with the vector control or A20/B7-1 variant.
Reduction ofB7-2 Expression in a mouse immunized with A20/B7-2. Only 4 animals of a total of 30 mice inoculated with the A20/B7-2-5 clone developed progressive tumours. One of these tumours was dissected and immunofluorescence staining performed. Figure 11 demonstrates that the level of B7-2 expressiori in the tumour cells is a little different from the endogenous level expressed by the parental cells, i.e., the level of B7-2 had decreased from that seen in the clone A20/B7-2-5. Although all the clones employed in the above experiments were cloned by limiting dilution it is unclear whether these tumour cells were wild-type contaminants in the A20/B7-2-5 inoculum or whether B7-2 down regulation occurred in vivo. These results suggest that a certain threshold level of B7-2 is necessary to mediate an anti-tumour imniune response and that in some situations tumour cells with lower levels of B7-2 expression may escape this anti-tumour response.
B7-2 modified tumour cells enhance survival in a minimal disease tumour model.
To mimic the clinical setting of established disease, animals were injected with parental cells on the left flank and 24 h later, animals were inoculated with 105 tumour variants on the opposite (i.e., right ) flank. Because profound anti-tumour immune responses were observed when A20 had been genetically modified with B7-1/mIL-12, comparisons of these variants in an established disease model were conducted.
Sixty-eight days post injection of the variant lines, 20% (2/10) of the mice remained alive in the vector control groups, 40 % survived in the A20/B7-l group, 30% survived in the mIL-12 group, 40 % survived in the B7-1 /mIL-12 group, and 70 % survived in the B7-2 group.
Moreover, the surviving animals injected with the A20/B7-2 variant were tumour free and had statistical increases in survival when compared to either the A20/pRc/CMV
(p<0.006) or A20/pRc/pac (p<0.02 - vector control groups (Fig. 12). In addition, a statistically significant increase in survival (p<0.072) was approached in favour of the B7-2 group when the A20/mIL-12 group was compared to the animals injected with the A20/B7-2 variant. Although the survival curves were superior, statistical differences in survival were not observed when the A20/B7-2 animals were compared with the 1 /mIL-12 variant (p>0.1).
In a more stringent experiment, we inoculated 105 A20 parental cells in the left flank and four days later, these mice were inoculated in the opposite flank with either 105 A20/pRc/CMV vector control, A20/B7-2-5 clone, or A20/B7-2 bulk variant cell lines. In this experiment, the protective effect of B7-2 was not observed. The mice injected with the A20/pRc/CMV variant developed tumours on both flanks whereas the mice injected with the A20/B7-2 variants developed tumours only on the left flank. The statistical analyses indicated that there was no statistical increase in survival in any of the three groups (data not shown).
Taken together, in this minimal disease model, animals injected with A20/B7-2 variant demonstrate anti-tumour immune responses, that are just as potent or greater than anti-tumour responses induced with the A20/B 7-1 /mIL-12 or A20/mIL-12 groups.
However, there are limitations on the potency of this effect since effective anti-tumour immunity can only be achieved in a limited disease state.
Mechanisms by which A20/B7-2 mediates anti-tumour immune responses. To gain insight into the mechanism responsible for the observed anti-tumour activity elicited by tumour cells engineered with B7-2, T cell deficient BALB/c nu/nu mice were inoculated with 105 A20/pRc/CMV, A20/B7-1, or A20/B7-2 variants. By day twenty-nine post injection all animals were euthanized with tumours approximating 2 cm in diameter (Fig.
13). Unlike the results seen in immunocompetent mice no statistical differences in survival were seen between the animals injected with the B7-1 or B7-2 transfectants or the vector control (p>0.08). these data suggest that T cells are important in mediating the anti-tumour response produced by the variant expressing higher levels of B7-2.
Since nude mice contain normal or even elevated levels of NK cells, these data also imply that NK cells play a minimal role.
In addition, CTZ, assays as measured by DNA fragmentation were performed usinb animals that had been immunized with the A20/pRe/pac vector control, A20/B7-2 clones 3 or 5, or the A20/mIL-12 variant lines. Animals were also inununized with combinations of A20/B7,2 and A20/miL-12 to detezxnine if the IL- 12 would synergize with the B7-2 in the inoculum to elali:ance CTL activitv. As shown +ri FigurP
iA, at -A-- === --- ---=
effector to target ratio of 40_ 1, CTL lytic activity frorn animals injected with either the A20B7-2-3 or A20/B7-2-5 clone was 78.3% and 77.2% respectively which was comparable or greater to the CTL activity seen from rnice inimunized with the variants. Coin,jection with the IL-12 variant did not augment the activity seen with the B7-2 variants.
To determine which T cell subset was predominantly mediating the observed anti-tumour imrrxune response in vivo, CD41 and CD8' T cells wcre depleted using mAbs.
By day nineteen, 60% of the mice depleted of CD8+ T cells and inoculated with the A20B7-2 variant had developed palpable tumours (Fig. 15), while animals remained tumour $ee in the CD4+ depleted group (data not shown). Interestingly, when CD8} T
cell depletion was terminated on day thirty-two, the tumours which had developed in mice inoculated with the A20/87-2 variant began to regress and by day thirty-seven only one mouse had a palpable tum.our remaining (Fig. 15). Taken together the depletiozi data and the CTL lysis results suggest that CD8+ T cells play a major role in mediating the aaxti-tumour immune response.
Both CD8+or CD41 T cell depleted mice inoculated with the A20/B7-1/mIL-12 variant were still able to reject this variant confirming our previous experiments (Pizzoferrato et al., 1997) and suggesting that the meclianism of anti-tumour immurnity mediated via these tumour cells is multifactorial.
Lymphokine activated killer cell (LAK) cultures were prepared by incubating BALB/c nu/nu splenocytes in media containing IL-2 for 48 h to determine whether natural killer cells could be functioning to mediate anti-tumour immune responses observed with the A20/B7-2, A20/mIL-12 or A20/B7-1 /mIL- 12 transfectants. Figure 16 demonstrates that the parental A20 cell line is a target for lysis by LAK cells. Differences in killing by these LAK cells were not observed between the parental and transfectant lines.
However, when the A20 cell variants were injected intravenously into BALB/c nu/nu mice and NK
cells were isolated from immunized mice, differences in killing capacity were detected.
Figure 17 indicates that the intravenously injected IL- 12 variants were capable of augmenting the natural killer cell killing of A20 parental cells when compared to the A20/B7-2 and vector control variants. These data illustrate that although the A20 is an effective target for killing by natural killer cells, the IL- 12 variants can augment the killing capacity. Moreover, although the A20/B7-2 variant elicits potent anti-tumour immune responses, natural killer cells play a minimal role in the observed augmented immune response.
Discussion for B7-2 Effective immune responses in murine tumour models have been elicited by introducing the costimulatory molecules, B7-1 or B7-2 (Chen et al., 1992;
Townsend et al., 1993; Yang et al., 1995; Gajewski et al., 1996; Martin-Fontecha et al., 1996). The relative effectiveness of B7-1 or B7-2 in generating anti-tumour immunity may depend upon the inherent immunogenicity of the tumour. In recent studies, B7-2 modified tumour cells induced superior anti-tumour responses in non-immunogenic tumours while B7-1 was just as effective as B7-2 in immunogenic tumours (Martin-Fontecha et al., 1996). In contrast, Matulonis et al., reported more profound anti-tumour effects when B7-1 was introduced into a non-immunogenic murine myeloid leukemia model (Matulonis et al., 1996).
Unlike most solid tumours which do not express B7-1 or B7-2, immunostaining demonstrates that B cell malignancies express the costimulatory molecule B7-2 (Schultze et al., 1995; Xerri et al., 1997; Van Gool et al., 1997). Despite the expression of this potent costimulator as well as MHC I and MHC II, freshly isolated patient samples of follicular lymphoma cannot generate efficient T cell responses (Schultze et al., 1995).
Only when stimulated via the CD40-L can these cells stimulate T cells in an allo MLR
(Schultze et al., 1995). Like most human B cell lymphomas, the non-immunogenic inurine B cell lymphoma does not express B7-1, but does express moderate levels of B7-2. Although the presence of B7-1 could indeed stimulate allogeneic CD4+ T
cells to secrete both IL-2 and IL-4, a profound in vivo anti-tumour immune response was not observed with B7-1 modified lymphoma cells (Pizzoferrato et al., 1997).
Despite the presence of moderate endogenous levels of B7-2 in the A20 cell line, only minimal levels of IL-4 are secreted from allogeneic T cells and 11-4 secretion is enhanced when B7-2 expression is augmented, suggesting that there may be a threshold of B7-2 expression required to induce T cell activation (Pizzoferrato et al., 1997). It is thus hypothesized that enhancement of B7-2 expression may lead to enhanced T cell activation and perhaps anti-tumour immune responses in a murine model of B cell lymphorna.
Indeed, when tumourigenicity studies were conducted with various A20 clones expressing only 2.5X the level of B7-2 than the parental cell line, immunogenicity was significantly augmented and these variants were rejected in syngene:ic hosts.
Moreover, the protected animals rejected challenge with the parental, unmodified cell line.
Interestingly, tumour escape variants were observed from the A20/B7-2 clone which had lost expression of B7-2 to the level seen in the parental clone. This observation is consistent with the idea that a critical threshold of B7-2 may be necessary to elicit anti-tumour immunity in tumour cells expressing endogenous B7-2. Recently, it has been shown that the effectiveness of low dose L-PAM chemotherapy is associated with the upregulation of B7-2 expression in the draining lymph nodes of MOPC-315 plasmacytoma tumour bearers. Since MOPC-315 cells from mice treated with L-PAM
express higher levels of B7-2 than B7-1, it is suggested that this higher expression may contribute to the importance of B7-2 in the MOPC-315 tumour system (Mokyr et al., 1998).
Strikingly, in a minimal disease tumour model, in which the parental line was inoculated 24 hr prior to vaccination with each of the variant lines, only animals treated with the A20/B7-2 clone had a statistically significant increase in survival when compared to the vector controls at the end of a 68 day time interval. In addition, 70% of these animals remained tumour free.
When the A20/B7-2 variants were inoculated 4 days post inoculation of the parental cell line, the anti-tumour protection was lost. A number of factors including the rapidity of tumour growth, or peripheral tolerance to tumour antigens may explain this observation. It has recently been shown that clonotypic CD4+ T cells initially expand a.nd lose their naive phenotype in mice injected with both A20/influenza hemagglutinin (HA) tumour cells and adoptively transferred anti-HA/I-Ed transgenic T cells.
These HA-specific T cells, however, have diminished proliferative responses to HA
peptide in vitro (Staveley-O'Carroll et al., 1998). This unresponsiveness was observed as early as 6 days post T cell transfer. Peripheral tolerance may occur in our mociel system as well. If this hypothesis is indeed correct, then subsequent restimulation with A20/B7-2 higher expressing variant tumour cell lines would result in similar unresponsiveness and these wuznals would be unable to mount effective anti-tumour responses. Moreover, T
cell anergy txtay result from low level 137 expression and selective binding to the negative regulator of iinmune responses, CTLA-4 (Perez et al., 1997).
In the first part of this description, it was shown that tumour cells modified with IL-12 in addition to B7-1 wm more effective in inducing anti-tClnl,our immunity than tuinoAr cells modified with either molecule alone. To investigate whether similar synergy exists between B7-2 and IL-12, mice were coinjected with both A20/B7-2 and A20/mIL-12 clones. In contrast to B7-1 and IL-12, synergy between B7-2 and IL-12 was not demonstrated in this murine model since the survival of animals inoculated with the $7-2 variants was not statistically different from that of mice coinjected with the and A20/mrL-12 variant clones.
Although a statistical difference was not noted between the A20/B7-2 and the A20/mIL-12 group in this minimal disease model, the survival of animals inoculated with A20/B7-2 approached statistical significance when compared to the A20/mIL-12 group.
Thi.s subgests that enhanced expression of B7-2 is equaE to or cnay be more effective than IS.-12 in generating anti-tumour irn.mune responses.
In several tumour models, enhanced anti-turnour immunitv elicited by B7-1 or B7-2 is primarily T cel.l mediated, although B7-1 has also been shown to enhance NK
lysis (Chambers et aL, 1996). While anRi-tumour immunity generated by B7-1 is primarily CD8+ T cell mediated (Townsend et al., 1993; Matulonis et al_; 1996), studies have demornstrated that CD4+ and/or CDS+ T cells are responsible for anti-tumour responses elicited by B7-2 (Martin.-p'ontecha et al., 1996; Matul.onis et aI., 1996). In this mtirine model of B cell lymphoma, the protection observed with the A20/B7-2 variants is inediated primarily by CD8+ T lymphocytes. Anti-tumour immunity was lost when A20/B7-2 variants were inoculated in nu/nu mice and more specifically in CD8 depleted but not CD4 depleted mice. In addition, enhanced CTL lysis as measured by DNA
fragmentation was seen in animals immunized with the B7-2 clones.
The mechanism of anti-tumour activity mediated by B7-2 modified A20 cells is different from that mediated by IL-12 or a combination of B7-1 and IL-12 modified lymphoma variants.
B7-1 can act as a triggering signal for NK lysis and may play a role in anti-tumour responses elicited by B7-1 transfected tumour cell lines (Yeh el al., 1995;
Wu. T.C. et al., 1995; Cavallo et al., 1995; Geldhof et al., 1995). For example, the 'T cell lymphoma, EL4, when transfected with B7-1, is efficiently killed by lymphokine-activated killer cells (LAK cells) (Chambers et al., 1996). In order to address whether LAK
cells or NK
cells could mediate the observed A20/B7-2 anti-tumour responses, LAK and NK
assays were performed with each of the variant cell lines. Lysis of A20 parental and variant lines by in vitro generated LAK cells was comparable. When intravenously injected into nu/nu mice, however, NK cells derived from mice immunized with the IL-12 or B7-12 variants were capable of augmented NK killing of the A20 target. This suggests that NK cells play a more important role in mediating the observed anti-tumour response in mice immunized with the A20/mIL- 12 or A20/B7-1/mIL-12 variant lines than with the B7-2 modified variants. This is consistent with studies demonstrating the effectiveness of IL-12 in enhancing NK cell mediated anti-tumour activity (Tahara et al., 1995).
Unlike Chambers et al. whose studies showed triggering of mouse LAK cells by (Chambers et al., 1996), these data do not show enhancement of LAK cell killing through the introduction of B7-1 or B7-2 into the A20 murine B cell lymphoma cell line. These data show that A20 is an NK-sensitive target and any increase in costimulation may not further enhance its susceptibility to NK mediated lysis. Moreover, despite the expression of MHC class I which acts as an NK inhibitory signal (Ljunggren et al., 1990;
Karre et al., 1995), the A20 parental and variant cell lines are sensitive to lysis by NK cells.
However, progressive tumours develop in mice inoculated with the A20 parental or A20/pRc/pac vector control variants suggesting that NK cell activity alone is not potent enough to prevent tumour outgrowth.
Although evidence indicates that B7-1 and B7-2 may have distinct functional roles, the relative effectiveness of either molecule in immunotherapeutic strategies aimed at treating cancer patients remains unclear. It is perhaps not surprising that different anti-tumour activity may be mediated by B7-1 or B7-2 costimulation. These molecules may differentially influence the immune response. For example, in vitro stimulation assays and evidence obtained by the murine model of experimental allergic encephalomyelitis (EAE) suggest that B7-2 stimulates naive CD4+ T cells to secrete IL-4, hence, skewing the immune response towards a ThO/Th2 phenotype (Kuchroo et al., 1995; Freeman et al., 1995). In contrast, B7-1 allows Th precursor cells to differentiate towards a Thi phenotype. These data indicate that introduction of B7-2 into the non-immunogenic B
cell lymphoma, A20, can enhance CTL activity and allow for profound anti-tumour immune responses. Taken together, anti-tumour immunity generated by B7-1 or B7-may depend upon both the immunogenicity of the tumour cell line as well as the cytokine environments induced by these costimulatory molecules.
This is particularly relevant to human B cell lymphomas which generally express moderate levels of B7-2 yet act as poor APCs (Schultze et al., 1995). This murine model niimics this phenomenon and demonstrates that a critical threshold of costimulatory activity may be necessary to activate "T cells and generate anti-tumour immune responses in B cell malignancies. These results may have important implications for immunogene therapy approaches aimed at treating B cell lymphomas.
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Claims (95)
1. An in vitro or ex vivo method of enhancing immunity against hematopoietic tumours in a mammal which has a hematopoietic tumour, said method comprising the steps of:
genetically modifying hematopoietic tumour cells from the same or a different mammal to express a cytokine and costimulatory molecule, in the mammal, wherein said cytokine and costimulatory molecule is IL-12 in combination with B7-1, and expressing said cytokine and costimulatory molecule in the presence of cells from said hematopoietic tumour, or, genetically modifying hematopoietic tumour cells from the same or a different mammal to express a costimulatory molecule, in the mammal, wherein said costimulatory molecule is B7-2, and expressing said costimulatory molecule in the presence of cells from said hematopoietic tumour.
genetically modifying hematopoietic tumour cells from the same or a different mammal to express a cytokine and costimulatory molecule, in the mammal, wherein said cytokine and costimulatory molecule is IL-12 in combination with B7-1, and expressing said cytokine and costimulatory molecule in the presence of cells from said hematopoietic tumour, or, genetically modifying hematopoietic tumour cells from the same or a different mammal to express a costimulatory molecule, in the mammal, wherein said costimulatory molecule is B7-2, and expressing said costimulatory molecule in the presence of cells from said hematopoietic tumour.
2. The method according to claim 1 wherein the step of genetically modifying further comprises the step of genetically modifying said hematopoietic tumour cells using at least one vector construct which directs the expression of a DNA
sequence, said at least one vector construct comprising:
IL-12 gene in combination with B7-2 gene; IL-12 gene in combination with B7-1 gene; B7-1 gene alone ; B7-2 gene alone; or IL-12 gene in combination with B7-1 gene in combination with B7-2 gene, to form genetically modified hematopoietic tumour cells, wherein said combination may be on one vector or on more than one vector.
sequence, said at least one vector construct comprising:
IL-12 gene in combination with B7-2 gene; IL-12 gene in combination with B7-1 gene; B7-1 gene alone ; B7-2 gene alone; or IL-12 gene in combination with B7-1 gene in combination with B7-2 gene, to form genetically modified hematopoietic tumour cells, wherein said combination may be on one vector or on more than one vector.
3. The method according to claim 2 wherein the at least one vector construct comprises a polynucleotide vector.
4. The method according to claim 2 wherein the at least one vector construct comprises a combination of a virus and a plasmid; or a virus and a polynucleotide; or a plasmid and a polynucleotide; or a virus and a plasmid and a polynucleotide.
5. The method according to claim 2 wherein the at least one vector construct comprises a virus.
6. The method according to claim 5 wherein the virus is fowlpox.
7. The method according to claim 5 wherein the virus is canarypox.
8. The method according to claim 5 wherein the virus is adenovirus.
9. The method according to claim 5 wherein the virus is vaccinia virus.
10. The method according to claim 5 wherein the virus is swine pox virus or polio virus.
11. The method according to claim 5 wherein the virus is a retrovirus.
12. The method according to claim 2 wherein the at least one vector construct comprises a plasmid.
13. The method according to claim 12 wherein the plasmid is a DNA plasmid comprising a promoter which allows for the expression of the gene or combination of genes.
14. The method according to claim 13 wherein the plasmid is pRc/CMV.
15. The method according to any one of claims 1 to 14 wherein the hematopoietic tumour is a leukemia, lymphoma, or myeloma.
16. The method according to claim 15 wherein the leukemia is acute lymphoblastic leukemia (ALL), acute myelocytic leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL) or juvenile myelo-monocytic leukemia (JMML).
17. The method according to claim 15 wherein the lymphoma is B-Cell Burkitt's lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Ki-1 positive anaplastic large cell lymphoma, T cell lymphoma or histiocytic lymphoma.
18. The method according to claim 15 wherein the myeloma is a multiple myeloma.
19. The method according to any one of claims 1 to 18 wherein the genetically modified tumour cells are present at the same site as a tumour burden.
20. The method according to any one of claims 1 to 18 wherein the genetically modified tumour cells are present at a site which is different from a tumour burden.
21. The method according to any one of claims 1 to 18 wherein the genetically modified tumour cells are present at a site which is distant from a tumour burden.
22. The method according to any one of claims 19, 20, or 21 wherein said tumour burden is defined as a minimal disease state.
23. The method according to any one of claims 1 to 22 wherein the genetically modified tumour cells are present in proximity to the hematopoietic tumour.
24. An in vitro or ex vivo method of enhancing immunity against hematopoietic tumours in a mammal which has a hematopoietic tumour, said method comprising the steps of:
genetically modifying hematopoietic tumour cells from the same or a different mammal to express a cytokine and costimulatory molecule, in the mammal, wherein said cytokine and costimulatory molecule is IL-12 in combination with B7-1, and expressing said cytokine and costimulatory molecule, in vitro or ex vivo in the presence of immune cells, said immune cells comprising antigen presenting cells, T cells, and/or NK cells, in the presence of naturally occurring mammalian hematopoietic tumour cells, or genetically modifying hematopoietic tumour cells from the same or a different mammal to express a costimulatory molecule, in the mammal, wherein said costimulatory molecule is B7-2, and expressing said costimulatory molecule, in vitro or ex vivo, in the presence of immune cells, said immune cells comprising antigen presenting cells, T cells, and/or NK cells, in the presence of naturally occurring mammalian hematopoietic tumour cells, and waiting until lysis or activation of cell death processes of said naturally occurring mammalian hematopoietic tumour cells occurs.
genetically modifying hematopoietic tumour cells from the same or a different mammal to express a cytokine and costimulatory molecule, in the mammal, wherein said cytokine and costimulatory molecule is IL-12 in combination with B7-1, and expressing said cytokine and costimulatory molecule, in vitro or ex vivo in the presence of immune cells, said immune cells comprising antigen presenting cells, T cells, and/or NK cells, in the presence of naturally occurring mammalian hematopoietic tumour cells, or genetically modifying hematopoietic tumour cells from the same or a different mammal to express a costimulatory molecule, in the mammal, wherein said costimulatory molecule is B7-2, and expressing said costimulatory molecule, in vitro or ex vivo, in the presence of immune cells, said immune cells comprising antigen presenting cells, T cells, and/or NK cells, in the presence of naturally occurring mammalian hematopoietic tumour cells, and waiting until lysis or activation of cell death processes of said naturally occurring mammalian hematopoietic tumour cells occurs.
25. The method according to claim 24 wherein the expression of cytokine and costimulatory molecule in vitro or ex vivo comprises introducing at least one vector construct which directs the expression of a DNA sequence, said at least one vector construct comprising:
IL-12 gene in combination with B7-2 gene; IL-12 gene in combination with B7-1 gene; B7-1 gene alone ; and B7-2 gene alone; or IL-12 gene in combination with B7-1 gene in combination with B7-2 gene, into the vicinity of the hematopoietic tumour, wherein said combination may be on one vector or on more than one vector.
IL-12 gene in combination with B7-2 gene; IL-12 gene in combination with B7-1 gene; B7-1 gene alone ; and B7-2 gene alone; or IL-12 gene in combination with B7-1 gene in combination with B7-2 gene, into the vicinity of the hematopoietic tumour, wherein said combination may be on one vector or on more than one vector.
26. The method according to claim 24 wherein the step of genetically modifying further comprises the step of genetically modifying said hematopoietic tumour cells using at least one vector construct which directs the expression of a DNA
sequence, said at least one vector construct comprising:
IL-12 gene in combination with B7-2 gene; IL-12 gene in combination with B7-1 gene; B7-1 gene alone ; B17-2 gene alone; or IL-12 gene in combination with B7-1 gene in combination with B7-2 gene, to form genetically modified hematopoietic tumour cells, wherein said combination may be on one vector or on more than one vector.
sequence, said at least one vector construct comprising:
IL-12 gene in combination with B7-2 gene; IL-12 gene in combination with B7-1 gene; B7-1 gene alone ; B17-2 gene alone; or IL-12 gene in combination with B7-1 gene in combination with B7-2 gene, to form genetically modified hematopoietic tumour cells, wherein said combination may be on one vector or on more than one vector.
27. The method according to claim 25 or 26 wherein the at least one vector construct comprises a polynucleotide vector.
28. The method according to claim 25 or 26 wherein the at least one vector construct comprises a combination of a virus and a plasmid; or a virus and a polynucleotide; or a plasmid and a polynucleotide; or a virus and a plasmid and a polynucleotide.
29. The method according to claim 25 or 26 wherein the at least one vector construct comprises a virus.
30. The method according to claim 29 wherein the virus is fowlpox.
31. The method according to claim 29 wherein the virus is canarypox.
32. The method according to claim 29 wherein the virus is adenovirus.
33. The method according to claim 29 wherein the virus is vaccinia virus.
34. The method according to claim 29 wherein the virus is swine pox or polio virus.
35. The method according to claim 29 wherein the virus is a retrovirus.
36. The method according to claim 25 or 26 wherein the at least one vector construct comprises a plasmid vector.
37. The method according to claim 36 wherein the plasmid is a DNA plasmid comprising a promoter which allows for the expression of the gene or combination of genes.
38. The method according to claim 37 wherein the plasmid is pRc/CMV.
39. The method according to any one of claims 24 to 38 wherein the hematopoietic tumour is a leukemia, lymphoma, or myeloma.
40. The method according to claim 39 wherein the leukemia is acute lymphoblastic leukemia (ALL), acute myelocytic leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL) or juvenile myelo-monocytic leukemia (JMML).
41. The method according to claim 39 wherein the lymphoma is B-Cell Burkitt's lymphoma, Hodgkin's lymphomas, non-Hodgkin's lymphomas, Ki-1 positive anaplastic large cell lymphomas, T cell lymphomas or histiocytic lymphomas.
42. The method according to claim 39 wherein the myeloma is a multiple myeloma.
43. The method according to any one of claims 24 to 42 wherein the genetically modified tumour cells are present at the same site as a tumour burden.
44. The method according to any one of claims 24 to 42 wherein the genetically modified tumour cells are present at a site which is different from a tumour burden.
45. The method according to any one of claims 24 to 42 wherein the genetically modified tumour cells are present at a site which is distant from a tumour burden.
46. The method according to any one of claims 43, 44, and 45 wherein said tumour burden is defined as minimal disease state.
47. A vaccine for enhancing immunity to hematopoietic tumours in a mammal which has a hematopoietic tumour, said vaccine comprising genetically modified hematopoietic tumour cells of a mammal, wherein said tumour cells are genetically modified to express a cytokine and costimulatory molecule wherein said cytokine and costimulatory molecule is IL-l2 in combination with B7-1, or wherein said tumour cells are genetically modified to express a costimulatory molecule wherein said costimulatory molecule is B7-2.
48. The vaccine according to claim 47 wherein the vaccine comprises live, replicating cells.
49. The vaccine according to claim 47 wherein the vaccine comprises non-replicating cells.
50. The vaccine according to any one of claims 47, 48 or 49 wherein the genetically modified hematopoietic tumour cells further comprise hematopoietic tumour cells transduced, transfected or infected using at least one vector construct which directs the expression of a DNA sequence, said at least one vector construct comprising:
IL-12 gene in combination with B7-2 gene; IL-12 gene in combination with B7-1 gene; B7-1 gene alone ; B7-2 gene alone; or IL-12 gene in combination with B7-1 gene in combination with B7-2 gene, wherein said combination may be on one vector or on more than one vector.
IL-12 gene in combination with B7-2 gene; IL-12 gene in combination with B7-1 gene; B7-1 gene alone ; B7-2 gene alone; or IL-12 gene in combination with B7-1 gene in combination with B7-2 gene, wherein said combination may be on one vector or on more than one vector.
51. The vaccine according to claim 50 wherein the at least one vector construct comprises a polynucleotide vector.
52. The vaccine according to claim 50 wherein the at least one vector construct comprises a combination of a virus and a plasmid; or a virus and a polynucleotide; or a plasmid and a polynucleotide; or a virus and a plasmid and a polynucleotide.
53. The vaccine according to claim 50 wherein the at least one vector construct comprises a virus.
54. The vaccine according to claim 53 wherein the virus is fowlpox.
55. The vaccine according to claim 53 wherein the virus is canarypox.
56. The vaccine according to claim 53 wherein the virus is adenovirus.
57. The vaccine according to claim 53 wherein the virus is vaccinia virus.
58. The vaccine according to claim 53 wherein the virus is swine pox virus or polio virus.
59. The vaccine according to claim 53 wherein the virus is a retrovirus.
60. The vaccine according to claim 50 wherein the at least one vector construct comprises a plasmid.
61. The vaccine according to claim 60 wherein the plasmid is a DNA plasmid comprising a promoter which allows for the expression of the gene or combination of genes.
62. The vaccine according to claim 61 wherein the plasmid is pRc/CMV.
63. The vaccine according to any one of claims 47 to 62 wherein the hematopoietic tumour is a leukemia, lymphoma, or myeloma.
64. The vaccine according to claim 63 wherein the leukemia is acute lymphoblastic leukemia (ALL), acute myelocytic leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL) or juvenile myelo-monocytic leukemia (JMML).
65. The vaccine according to claim 63 wherein the lymphoma is B-Cell Burkitt's lymphoma, Hodgkin's lymphomas, non-Hodgkin's lymphomas, Ki-1 positive anaplastic large cell lymphomas, T cell lymphomas or histiocytic lymphomas.
66. The vaccine according to claim 63 wherein the myeloma is a multiple myeloma.
67. A pharmaceutical composition for use as a vaccine for enhancing immunity to hematopoietic tumours in a mammal which has a hematopoietic tumour, said pharmaceutical composition comprising, genetically modified hematopoietic tumour cells of a mammal wherein said tumour cells are genetically modified to express a cytokine and costimulatory molecule wherein said cytokine and costimulatory molecule is IL-12 in combination with B7-1 or genetically modified hematopoietic tumour cells of a mammal wherein said tumour cells are genetically modified to express a co-stimulatory molecule wherein said costimulatory molecule is B7-2;
together with a pharmaceutically acceptable carrier or diluent.
together with a pharmaceutically acceptable carrier or diluent.
68. The composition according to claim 67 wherein said genetically modified hematopoietic tumour cells further comprise hematopoietic tumour cells transduced, transfected or infected using at least one vector construct which directs the expression of a DNA sequence, said at least one vector construct comprising:
IL-12 gene in combination with B7-2 gene; IL-12 gene in combination with B7-1 gene; B7-1 gene alone ; B7-2 gene alone; or IL-12 gene in combination with B7-1 gene in combination with B7-2 gene, wherein said combination may be on one vector or on more than one vector.
IL-12 gene in combination with B7-2 gene; IL-12 gene in combination with B7-1 gene; B7-1 gene alone ; B7-2 gene alone; or IL-12 gene in combination with B7-1 gene in combination with B7-2 gene, wherein said combination may be on one vector or on more than one vector.
69. The composition according to claim 68 wherein the at least one vector construct comprises a polynucleotide vector.
70. The composition according to claim 68 wherein the at least one vector construct comprises a combination of a virus and a plasmid; or a virus and a polynucleotide; or a plasmid and a polynucleotide; or a virus and a plasmid and a polynucleotide.
71. The composition according to claim 68 wherein the at least one vector construct comprises a virus.
72. The composition according to claim 71 wherein the virus is fowlpox.
73. The composition according to claim 71 wherein the virus is canarypox.
74. The composition according to claim 71 wherein the virus is adenovirus.
75. The composition according to claim 71 wherein the virus is vaccinia virus.
76. The composition according to claim 71 wherein the virus is swine pox virus or polio virus.
77. The composition according to claim 71 wherein the virus is a retrovirus.
78. The composition according to claim 68 wherein the at least one vector construct comprises a plasmid.
79. The composition according to claim 78 wherein the plasmid is a DNA plasmid comprising a promoter which allows for the expression of the gene or combination of genes.
80. The composition according to claim 79 wherein the plasmid is pRc/CMV.
81. The composition according to any one of claims 67 to 80 wherein the hematopoietic tumour is a leukemia.
82. The composition according to any one of claims 67 to 80 wherein the hematopoietic tumour is a lymphoma.
83. The composition according to any one of claims 67 to 80 wherein the hematopoietic tumour is a myeloma.
84. The composition according to claim 81 wherein the leukemia is acute lymphoblastic leukemia (ALL), acute myelocytic leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL) or juvenile myelo-monocytic leukemia (JMML).
85. The composition according to claim 82 wherein the lymphoma is B-Cell Burkitt's lymphoma, Hodgkin's lymphomas, non-Hodgkin's lymphomas, Ki-1 positive anaplastic large cell lymphomas, T cell lymphomas or histiocytic lymphomas.
86. The composition according to claim 83 wherein the myeloma is a multiple myeloma.
87. The pharmaceutical composition for use as a vaccine according to any one of claims 67 to 86 for reducing hematological tumour burden in a mammal.
88. The pharmaceutical composition for use as a vaccine according to any one of claims 67 to 86 wherein said vaccine is suitable for administration intravenously, intradermally, orally, intranasally, subcutaneously, intranodally, intralymphatically, or intrathecally.
89. The pharmaceutical composition for use as a vaccine according to claim 88 wherein said vaccine is suitable for administration at the site of the hematological tumour.
90. The pharmaceutical composition for use as a vaccine according to claim 88 wherein said vaccine is suitable for administration at a site different from the hematological tumour.
91. The pharmaceutical composition for use as a vaccine according to claim 88 wherein said vaccine is suitable for administration at a site distant from the hematological tumour.
92. The pharmaceutical composition according to any one of claims 67 to 86 in the preparation of a medicament or a medicine wherein said medicine or medicament is suitable for administration intravenously, intradermally, orally, intranasally, subcutaneously, intranodally, intralymphatically, or intrathecally.
93. The pharmaceutical composition in the preparation of a medicament or a medicine according to claim 92 wherein said medicine or medicament is suitable for administration at the site of the hematological tumour.
94. The pharmaceutical composition in the preparation of a medicament or a medicine according to claim 92 wherein said medicine or medicament is suitable for administration at a site different from the hematological tumour.
95. The pharmaceutical composition in the preparation of a medicament or a medicine according to claim 92 wherein said medicine or medicament is suitable for administration at a site distant from the hematological tumour.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002253790A CA2253790C (en) | 1998-11-25 | 1998-11-25 | Methods of enhancing anti-tumour immunity in a mammal |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002253790A CA2253790C (en) | 1998-11-25 | 1998-11-25 | Methods of enhancing anti-tumour immunity in a mammal |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2253790A1 CA2253790A1 (en) | 2000-04-24 |
| CA2253790C true CA2253790C (en) | 2009-05-05 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002253790A Expired - Lifetime CA2253790C (en) | 1998-11-25 | 1998-11-25 | Methods of enhancing anti-tumour immunity in a mammal |
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| Country | Link |
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| CA (1) | CA2253790C (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008134879A1 (en) | 2007-05-04 | 2008-11-13 | University Health Network | Il-12 immunotherapy for cancer |
| US8568709B2 (en) | 2008-03-20 | 2013-10-29 | University Health Network | Thymidylate kinase fusions and uses thereof |
| CN114752628B (en) * | 2022-05-30 | 2023-06-30 | 四川大学华西医院 | Construction method and application of C57BL/6 mouse multiple myeloma model |
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1998
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| CA2253790A1 (en) | 2000-04-24 |
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