KR20050000376A - Activation of Tumor-Reactive Lymphocytes via Antibodies or Genes Recognizing CD3 or 4-1BB - Google Patents

Abstract

The present invention includes, for example, culturing PBMCs from a patient suffering from cancer with autologous tumor cells and magnetic beads capable of activating T lymphocytes through CD3 and CD28 or CD40 or CD28 and CD40. An improved method of producing T cells in vitro is provided. The present invention also provides genes encoding anti-CD3 or anti-4-1BB scFv, and provides a method of transfecting neoplasia or normal cells to express these genes on the surface. Such genes and transfected tumor cells are useful compositions for the induction of tumor-destructive immune responses.

Description

Activation of Tumor-Reactive Lymphocytes via Antibodies or Genes Recognizing CD3 or 4-1BB}

Related Applications

This patent application claims the benefit of US Patent Application No. 10 / 107,991, filed March 26, 2002.

Statement on federally sponsored research

Portions of this study are funded by the National Institutes of Health, and the US government may have certain rights in the invention.

The following description includes information that may be useful in understanding the present invention. It is not permissible to use any of the information provided herein as prior art, or to use any publications or documents that relate to the invention described or claimed herein or specifically or implicitly mentioned as prior art.

Human tumors express various tumor antigens, most of which are present in some normal tissues, although at low levels (Hellstrom and Hellstrom, Adv Cancer Res, 12, 167-223, 1969), Cheever et al., Immunol Rev, 145, 33-59, 1995, Finn et al., Immunol Rev, 145, 61-89, 1995, Boon et al., Immunol Today, 18, 267-8, 1997, Hellstrom and Hellstrom, Handbook of Experimental Pharmacology, Vaccines (Chapter 17), 463-478, 1999]. Many of these antigens are immunogenic in tumor-bearing hosts. For example, IgG antibodies against various tumor-associated antigens can be detected by SEREX technology and described in Old and Chen, J. Exp. Med., 187, 1163-1167, 1998], T cells that recognize tumor antigens can be demonstrated using tetramers, as well as tumor-selective T [Cassian et al., J. Immunol., 162, 1999]. This can be demonstrated by the ability to generate cell clones in vitro (Boon, Coulie et al., Immunol Today, 18, 267-8, 1997). This is due to administration of in vitro expanded immune T lymphocytes [Rosenberg, Biologic Therapy of Cancer (Chapter 19), 487, 1995] and tumor vaccination for treatment (Nestle et al., Nat Med, 4, 328-32, 1998, Rosenberg et al., Nat Med, 4, 321-7, 1998, Greenberg, Adv Immunol, 49, 281-355, 1991, Elief and Kast, Immunol Rev, 145, 167-77, 1995], [Pardoll, Curr Opin Immunol, 8, 619-21, 1996], [Hellstrom and Hellstrom, Handbook of Experimental Pharmacology, Vaccines (Chapter 17), 463-478, 1999]). To provide opportunities.

Mouse tumors provide a useful model for developing more effective immunotherapy. Although specific experimental manipulations are required to obtain an effective immune response against most tumors of spontaneous origin, mouse tumors express targets for tumor destructive immune responses (Greenberg, Adv Immunol, 49, 281-355, 1991). Kerr and Mule ', J. Leuko. Biol., 56, 210-214, 1994, Cheever, Disis et al., Immunol Rev, 145, 33-59, 1995, Finn, Jerome et al ., Immunol Rev, 145, 61-89, 1995, Melief and Kast, Immunol Rev, 145, 167-77, 1995, Pardoll, Curr Opin Immunol, 8, 619-21, 1996, Boon, Coulie et al., Immunol Today, 18, 267-8, 1997, Hellstrom and Hellstrom, Handbook of Experimental Pharmacology, Vaccines (Chapter 17), 463-478, 1999).

T lymphocytes (CD8 + and CD4 +) play a key role in generating tumor-destructive responses (and usually also performing tumor-destructive responses), but NK cells, antibodies and macrophages also contribute to this (Hellstrom and Hellstrom). , Adv Cancer Res, 12, 167-223, 1969, Greenberg, Adv Immunol, 49, 281-355, 1991, Melief and Kast, Immunol Rev, 145, 167-77, 1995, Hellstrom and Hellstrom , Handbook of Experimental Pharmacology, Vaccines (Chapter 17), 463-478, 1999]. Antigen presentation is usually carried out by dendritic cells (DCs) differentiated from stem cells in the bone marrow and monocytes in the blood (Huang et al., Science, 264, 961-5, 1994), but in certain situations the tumor cells themselves. (Chen et al., Cell, 71, 1093-102, 1992, Schenberger et al., Cancer Res, 58, 3094-100, 1998). Facilitating tumor antigen presentation by DC is very important for obtaining effective tumor immunity, and recently there is data indicating that this may enable more effective therapies for certain human cancers (eg, Kugler et al., Nature Medicine, 6, 332-, 2000). Most tumor rejections require a combination of CD8 + T lymphocytes and in vitro cytotoxic T lymphocyte (CTL) activity and lymphokine-producing T helper cells [Hellstrom and Hellstrom, Handbook of Experimental Pharmacology, Vaccines (Chapter 17), 463-478, 1999]. Both Th1 and Th2 responses may work advantageously (Rodolfo et al., T Immunol, 163, 1923-8, 1999), but the Th1 response plays a dominant role in immune destruction of tumors [Hu et al., J Immunol , 161, 3033-41, 1998].

In order to elicit an immune response, in particular, co-stimulation by CD80 on antigen-presenting cells (APCs) and / or CD86 and CD28 on T lymphocytes is required (Schwartz, Cell, 57). , 1073-81, 1989, June et al., Immunol Today, 11, 211-6, 1990, Linsley and Ledbetter, Annu Rev Immunol, 11, 191-212, 1993). This allows for the continuous production of IL-2, IFN-γ and other lymphokines necessary for amplifying immune responses (Thompson et al., Proc Natl Acad Sci USA, 86, 1333-7, 1989), encoding lymphokines Similar objectives are achieved by using tumor cells transfected with genes (Pardoll, Curr Opin Immunol, 8, 619-21, 1996). In the absence of a second signal through CD28, exposure of the T-cell receptor (TCR) to the antigen does not induce an immune response and may even induce no response. Most tumors do not express CD80 or CD86 (Chen, Ashe et al., Cell, 71, 1093-102, 1992, Yang et al., J Immunol, 154, 2794-800, 1995), Effective immunity is not induced until the antigen reaches the tumor-draining lymph nodes, is received, processed, and is presented by a DC expressing CD80 and CD86 (Huang, Golumbek et al., Science, 264, 961-5, 1994, Yang et al., J Immunol, 158, 851-8, 1997). This may explain why tumors often "sneak through" the immune system until there is an established tumor mass. CD80 and CD86 bind not only to CD28 but also to CTLA4 on activated T cells with much higher avidity. This binding with CTLA4 induces a negative signal that can terminate the immune response (Thompson, Lindsten et al., Proc Natl Acad Sci USA, 86, 1333-7, 1989, Walunas et al., Immunity, 1 , 405-13, 1994, Krummel and Allison, J Exp Med, 183, 2533-40, 1996, Teach et al., Science, 271, 1734-6, 1996, Walunas et al., J Exp Med, 183, 2541-50, 1996, [Allison et al., Novartis Found Symp, 215, 92-8, 1998]), which is a method involving CTLA4, while the method involving CD28 has therapeutic advantages. Indicates no.

Despite the possibility of inducing anti-tumor immunity, the immune system is relatively ineffective in destroying established tumors. Immune responses, such as those induced after delivery of conventional tumor vaccines or immune T cells with in vitro anti-tumor activity, can hardly destroy more than millions of tumor cells. Several escape mechanisms have been identified that may be involved in protecting tumors from immunological attacks (Hellstrom and Hellstrom, Handbook of Experimental Pharmacology, Vaccines (Chapter 17), 463-478, 1999), Kiessling et al., Cancer Immunol. Immunother., 48, 353-362, 1999). These include the loss of tumor epitopes [Maeurer et al., J Clin Invest, 98, 1633-41, 1996] and / or the loss of MHC class I molecules that can present such epitopes in CTL ([Restifo, 1993]. ], [Maeurer, 1996], [Hellstrom, 1997], [Garrido, 1997], [Johnsen, 1998]) and removal of tumor-reactive lymphocytes by apoptosis ([Hahne et al., Science, 274, 1363-1366, 1996, Shiraki et al., Proc Natl Acad Sci USA, 94, 6420-5, 1997, Bennett et al., J Immunol, 160, 5669-75, 1998, Kume et al., Int J Cancer, 84, 339-43, 1999, Chappell and Restifo, Cancer Immunol Immunother, 47, 65-71, 1998). However, tumors that can present immunogenic tumor antigens and do not induce apoptosis of reactive lymphocytes typically deviate from immune control. This is due to various "blocking factors" produced by the tumor, host, or both, with or without epitope selectivity, which act directly on T cells or indirectly through APC. These include TGF-β, prostaglandins, NO, as well as soluble tumor antigens and immune complexes (Hellstrom and Hellstrom, Adv Immunol, 18, 209-77, 1974), Kehrl et al., J Exp Med, 163 , 1037-50, 1986, Sulitzeanu, Adv Cancer Res, 60, 247-267, 1993, Kiessling et al., Springer Semin.Immunopathol., 18, 227-242, 1996, Kiessling, Wasserman et al., Cancer Immunol. Immunother., 48, 353-362, 1999]. Tumor-destructive Th1 response [Hu, Urba et al., J Immunol, 161, 3033-41, 1998] to the Th2 response [Fiorentino et al., J Exp Med, 170, 2081-95, 1989] Downregulation may occur by antigen released from cells alone or by immune complexes combining the antigens and antibodies, accompanied by the production of TGF-β, which may be of second importance in the production of Th2 lymphokines such as IL-10. (Wilbanks et al., Eur J Immunol, 22, 165-73, 1992, D'Orazio and Niederkorn, J Immunol, 160, 2089-98, 1998). Downregulation may be due to the interaction between CTLA4 on activated T lymphocytes and CD80 / CD86 on activated T cells or APC (Leach, Krummel et al., Science, 271, 1734-6, 1996), [ Allison, Chambers et al., Novartis Found Symp, 215, 92-8, 1998]), and may be mediated by TGF-β [Chen et al., J Exp Med, 188, 1849-57, 1998]. Downregulation of macrophages to produce NO and prostaglandins has also been identified [Kiessling, Wasserman et al., Cancer Immunol. Immunother., 48, 353-362, 1999].

Inhibition of T cell responsiveness reports that the T cell signaling mechanism often disappears in tumor-infiltrating T lymphocytes (Mizoguchi et al., Science, 258, 1795-8, 1992), Nakagomi et al., Cancer Res, 53 , 5610-5612, 1993, Kiessling, Wasserman et al., Cancer Immunol. Immunother., 48, 353-362, 1999) and can be recovered when these lymphocytes are removed from the body.

For example, reports on the use of T cell activating molecule 4-1BB to treat mouse tumors (Melero et al., Nat Med, 3, 682-5, 1997), Melero et al., Eur J Immunol, 28, 1116-21, 1998], even in large tumors, there are several situations where rejection is caused by the immune system. However, the most shocking example is probably the observation that some patients with large metastases originating from cancer cells have recovered completely after the release of immunosuppression during the early days of body transplanted kidney transplantation (Wilson et al., N Engl). J Med, 278, 479-83, 1968, Matt et al., Transplantation, 9, 71-4, 1970).

The cell surface molecule 4-1BB is expressed in activated T cells but not in naïve T cells (DeBenedette et al., J Exp Med, 181, 985-92, 1995), Shuford et al., J Exp Med, 186, 47-55, 1997]. The involvement of 4-1BB can amplify the immune response that has already been induced. Exposure to anti-4-1BB MAb results in proliferation of antigen-activated CD8 + T lymphocytes with CTL activity, production / release of IFN-γ and other cytokines of type Th1 (IL-2, TNF-α) and apoptosis Has been reported to stimulate T cell protection against (Hurtado et al., J Immunol, 158, 2600-9, 1997), Kim et al., Eur J Immunol, 28, 881-90, 1998, Natoli et al., Biochem Pharmacol, 56, 915-20, 1998, Takahashi et al., J Immunol, 162, 5037-40, 1999, Tsushima et al., Exp Hematol, 27, 433-40, 1999]. 4-1BB is also known to have an immunomodulatory effect involving NK1.1 cells (Melero et al., Cell Immunol, 190, 167-72, 1998).

In addition, it has been reported that MAb against 4-1BB is active against well-established (approximately 10 mm diameter) tumors, including tumors with low immunogenicity in mice and treated with anti-4-1BB MAb. Lymphocytes of mice produce CD8 + CTLs with increased cytolytic activity (Melero, Shuford et al., Nat Med, 3, 682-5, 1997). Exposure of lymphocytes to tumor cells incorporating 4-1BB ligand (4-1BBL) that is transfected and binds to 4-1BB may significantly amplify the CD8 + T cell response, and 4-1BBL-transfected tumor cells It has therapeutic activity when used as a vaccine in mouse models. However, there is evidence that administration of anti-4-1BB MAb is more effective than vaccination with tumor cells expressing 4-1BBL. Antibody treatment is effective against non-immunogenic sarcoma Ag104 (Melero, Shuford et al., Nat Med, 3, 682-5, 1997), but in the case of Ag104 cells transfected and expressing 4-1BBL. Combination with CD80 expressing cells is required to obtain a therapeutic effect on the tumor [Melero, 1998].

An alternative approach to increasing tumor immunity may be to administer a dose of anti-CD3 MAb that will provide polyclonal T cell activation, including activation of any tumor-responsive lymphocyte clone, and several treatments using this approach. Success stories have been reported in studies using mouse models (Ellenhom et al., Science, 242, 569-71, 1988).

As indicated to deliver tumor immunity using lymphocytes to prevent transplanted cells from each neoplasia from outgrowth [Klein et al., Cancer Res, 20, 1561-1572, 1960]. Likewise, tumor-reactive T cells can be generated in vitro for in vivo use. Not long ago, rejection of established small tumors was reported after adoptive transfer of immune lymphocytes (Hellstrom et al., Transplantation Proc, 1, 90-4, 1969). Adoptive-delivered lymphocytes preferentially localize to immunized tumors [Mule 'et al., J. Immunol., 123, 600-606, 1979], which finds treatment of in vitro expanded tumor-infiltrating lymphocytes (TILs). Has been stimulated for use (Rosenberg, Biologic Therapy of Cancer (Chapter 19), 487, 1995). In some classes of patients, dramatic clinical responses have occurred, but the extent of treatment success has been observed in mice with tumors larger than a few mm in diameter (Greenberg, Adv Immunol, 49, 281-355, 1991), Chen, Ashe et. al., Cell, 71, 1093-102, 1992, Melief and Kast, Immunol Rev, 145, 167-77, 1995, Hellstrom and Hellstrom, Handbook of Experimental Pharmacology, Vaccines (Chapter 17), 463-478 , 1999), and often only moderate to both humans. This failure may be due to one or more of the “departure” mechanisms discussed above and / or intratumoral localization errors of the injected lymphocytes.

Therefore, improved methods are needed to produce tumor vaccines that elicit stronger immune responses and to generate T lymphocytes for cancer patient therapy.

Summary of the Invention

The present invention relates to improved methods for producing tumor-reactive T cells in vitro and related compositions for use in tumor vaccines and in vivo therapeutics.

In one method, for example, mononuclear lymphoid cells from peripheral blood or tumors are harvested from cancer patients and cultured with autologous tumor cells in the presence of immobilized antibodies specific for CD3 and CD28. Such culturing can be carried out over a period of 4 to 5 days, for example. Cells can be propagated to levels useful for treatment in, for example, 10 μl / ml IL-2 after removal of antibody immobilized beads. Such methods are useful for improving the production of tumor-reactive lymphocytes for cancer therapy. While not being bound by any particular theory or mechanism, the present invention is believed to work through the following factors: T lymphocytes with low CD3 expression levels are polyclonal activated to proliferate violently and form Th1-type lymphokines. It releases tumor antigens while rapidly destroying tumor cells. In addition, polyclonal T cell activation causes monocytes in culture to mature into dendritic cells, which continue to receive tumor-specific T cells, including CTLs, by accepting and processing dead tumor cells to present tumor antigens. It is believed to induce proliferation.

The present invention also provides genes encoding anti-CD3 or anti-4-1BB single chain Fv (scFv) molecules expressed on the tumor cell surface and cells transfected with these genes for in vivo cancer therapy. . Without being bound by any particular theory or mechanism, anti-CD3 scFv expression on the surface of tumor cells is polyclonal, as detected by rejection of "wild-type" cells (untransfected cells) from the same tumor. It is believed to induce T cell activation and tumor cell destruction to release tumor antigens and to promote metastasis to antigen-specific tumor immunity. In addition, anti-4-1BB scFv expression on tumor cell surfaces increases tumor-reactive T cells proliferation and / or protects these cells from apoptosis, leading to tumor-reactive lymphokines such as IFN-γ. By allowing them to be produced, it is believed to induce activation / proliferation of tumor-reactive T cells. Again, without being bound by any particular theory or mechanism, after transfection and immunization with tumor cells expressing anti-4-1BB scFv on the cell surface, mechanisms, including activation of NK cells and CD4 + T cells, from the same tumor It is believed to cause rejection of wild-type cells. Tumor cells expressing anti-4-1BB scFv on the cell surface are active in the therapy of established wild type tumors.

The present invention enables, for example, two new cancer therapy methods. First, the present invention activates "inhibited" lymphocytes, for example, by immobilized anti-CD3 / anti-CD28 / anti-CD40 (or anti-CD3 / CD28) beads, whereby they proliferate to produce Th1 lymphokine and It causes the susceptibility to inhibition by TGF-β. Without being bound by any particular theory or mechanism, it is believed that activated lymphocytes provide tumor antigens by destroying tumor cells while also inducing maturation of APCs. This method is believed to allow for the proliferation of tumor-reactive CD8 + and CD4 + T cells and NK cells that are more suitable for adoptive delivery to cancer patients over time. Second, the present invention provides genes or polynucleotides encoding, for example, anti-CD3 or anti-4-1BB scFv at the tumor cell surface, to effectively induce tumor-destructive immune responses against wild-type cells from the same tumor. Can be.

Other aspects of the invention will be set forth in the following claims, which are hereby incorporated by reference in their entirety.

The present invention relates to methods of producing tumor-reactive T cells, tumor vaccines and compositions related thereto, methods of making the vaccines and compositions, and methods of using the vaccines and compositions, for example for in vivo healing use.

While the invention is broad and not limited to any examples described herein or mentioned herein, various aspects of the invention may be better understood upon reference to the accompanying drawings.

In vitro proliferation of isolated tumor infiltrating lymphocytes (TILs) from patients with advanced ovarian carcinoma (OV44). Panel A shows proliferation of lymphocytes stimulated with control beads and autologous tumor cells. Panel B shows lymphocyte proliferation after stimulation with anti-CD3 / CD28 / CD40 conjugated beads and autologous tumor cells. Panel C shows lymphocyte proliferation after stimulation with anti-CD3 / CD28 / CD40 beads without addition of tumor cells.

2. Autologous tumor cells induce PBMC proliferation from patients suffering from colon carcinoma when combined with beads stimulating CD3 and CD28, but not in the case of allogeneic tumor cells. 2 shows peripheral blood lymphocyte proliferation from cancer patients 1C after 5 days of in vitro stimulation in the presence of autologous (1C) or allogeneic 4007 ovarian carcinoma cells. Stimulation was as follows: A = control beads; B = anti-CD28 / CD40 conjugated beads; C = anti-CD3 / CD28 conjugated beads; D = anti-CD3 / CD28 / CD40 conjugated beads; E = anti-CD3 / CD40 conjugated beads.

PBMCs from patients with colon carcinoma lyse autologous tumor cells in the presence of beads that stimulate CD3 and CD28 or both CD3 and CD28 and CD40 (4 hour Cr ⁵¹ release assay). FIG. 3 shows cell-mediated cytotoxicity of PBL isolated from Patient 1C, with autologous tumor cells and anti-CD3 / CD28 beads (A) or autologous tumor cells and anti-CD3 / CD28 / CD40 beads (B). After activating the PBL it was tested in the indicated target cells.

4. PBMCs from patients with head and neck carcinoma produce IFN-γ after incubation with autologous tumor cells and beads that stimulate CD3 and CD28. 4 shows IFN-γ produced by PBL from patient 1HN with advanced head and neck carcinoma. IFN-γ levels in the culture supernatants were measured at different time points after stimulation with anti-CD3 / CD28 alone (A), anti-CD3 / CD28 beads and autologous tumor cells (B) or control beads and autologous tumor cells (C). .

5. CD83 is expressed after stimulation with beads stimulating CD3 and CD28 in PBMCs from patients with colon carcinoma. 5 shows CD83 expression in PBMCs isolated from patients with advanced cancer (38C). Before stimulation (day 0) or with anti-CD3 / CD28 beads and after 1 day the cells were stained with anti-CD83 (conjugated directly to phycoerythrin (PE)).

Figure 6. The level of CD83 expressed in PBMCs from patients with colon carcinoma is higher when stimulated with CD3 and CD28 than two days after stimulation with CD3 and CD28 and CD40. As shown, FIG. 6 shows that PBMC from a patient with advanced cancer (38C) was not activated or activated 2 days after stimulation with anti-CD3 / CD28 / CD40 beads, or with anti-CD3 / CD28 beads It is activated after 2 days. Cells were stained simultaneously with fluorescein-conjugated anti-CD3 and PE-conjugated anti-CD83.

7. K1735 melanoma cells (500A2) transfected and expressing anti-CD3 scFv on the cell surface regress when compared to K1735 cells and wild type K1735 cells expressing murine CD80.

8. K1735-WT cells transplanted into syngeneic mice immunized repeatedly with K1735-500A2 cells regressed. C3H / HeN mice were immunized three times at 7-day intervals with K1735M2 / 500A2 cells (2 × 10 ⁶ cells / mouse), followed by subcutaneous injection of K1735M2 wild type cells at 1 × 10 ⁶ cells / mouse (blue lines). After C3H / HeN mice were immunized with PBS, K1735M2 wild type was also injected at the same dose (red line).

Figure 9. Sequence of anti-human CD3 scFv gene—ie, the sequence of anti-human CD3 scFv-hIgG1-CD80 ™ synthetic polynucleotide encoding the product expressed on the cell surface for use in transfection.

10. Expression of anti-human CD3 scFv on the surface of two human cell lines after retroviral gene transduction. Anti-human CD3 scFv (G19-4) was expressed on Reh and T51 cell line surfaces via retroviral gene transduction. Surface expression of the G19-4 scFv gene product was detected using fluorescein-conjugated anti-human IgG that detects IgG1 CH2 and CH3 domains contained in the gene product. Responses to wild-type cells in each panel are shown by dashed lines and responses with transfected cells are shown by solid lines.

11. Proliferation of human T cells co-cultured with human cell lines expressing anti-CD3 scFv on the surface. 11 shows that incubation of T cells with Reh or T51 cells expressing anti-CD3 scFv on the cell surface induces T cell proliferation, but not when wild type Reh or T51 cells are used. Proliferation was measured by ³ H-thymidine incorporation during the last 8 hours of 3 day culture.

12. Resting human PBMCs lyse cells from two human cell lines expressing anti-CD3 scFv on the surface. 12 shows that resting PBMCs rapidly kill Reh and T51 cells expressing anti-CD3 scFv on tumor cell surfaces, but not wild type Reh or T51 cells. ^{The 51} Cr-labeled cell lines were incubated with PBMCs for 3 hours at the indicated cell ratios for 8 hours and then the released ⁵¹ Crs were measured. Specific percent mortality was determined according to conventional formula ((experimental release-spontaneous release) / (maximum release-spontaneous release)).

13. K1735-500A2 cells expressing anti-CD3 scFv on the surface inhibited tumor formation from mixed K1735-WT cells when the ratio of K1735-500A2: WT cells was 1:10, resulting in a "bystander effect. effect) ". FIG. 13 shows that the mixture of K1735-500A2 cells and K1735-WT cells (ratio of 1:10) is inhibited from overgrowth in immunocompetent syngeneic (C3H) mice. K1735-WT cells alone were used to immunize or immunized by mixing K1735-WT cells in a ratio of 10: 1 between K1735-500A2 transfected and untransfected cells: transfected tumor cells. 2 × 10 ⁶ K1735-WT cells were mixed with 2 × 10 ⁵ K1735-500A2 cells and the cells were reversed subcutaneously by avoiding C3H mice. Tumor growth was monitored at 5 day intervals.

14. Splenocytes from mice immunized with K1735-500A2 cells proliferate when combined with irradiated K1735-WT cells, but not when combined with Ag104 cells.

15. Transplantation of K1735-1D8 cells into syngeneic mice results in rejection by a mechanism dependent on both CD4 + T cells and NK cells. FIG. 15 shows that when K1735-1D8 cells are transplanted into C3H mice, rejection occurs by CD4 + T cells and NK cell dependent mechanisms.

A. Structure of retroviral vector containing scFv DNA from anti-murine 4-1BB hybridoma 1D8.

B. 1D8 scFv expression on K1735-1D8 cell surface. PE conjugated F (ab ') from goat-anti-human Ig recognizing immunoglobulin tails (dark region) expressed on K1735-1D8 cells (not expressed in K1735-WT cells (solid line)) Detected by ₂ .

C. Growth Dynamics of K1735-1D8 (○) and K1735-WT (■) Cells in Natural Mice.

D. Growth of K1735-1D8 cells in mice deficient in CD4 + (■), CD8 (□), CD4 + and CD8 + (○) T cells or NK (Δ) cells and control (●) mice injected with purified rat IgG dynamics.

16. Immunization with K1735-1D8 cells protected the transplanted K1735-WT cells from overgrowth, but not with immunization with irradiated K1735-WT cells, which was due to mechanisms with memory and specificity. It is considered to be.

A. K1735-1D8 (○) or irradiated K1735-WT (□) cells were implanted subcutaneously or injected with PBS (■) to immunize mice (10 mice / group) twice at 10 day intervals. Ten days after the last immunization, these mice were injected with K1735-WT cells. Mice immunized against K1735-1D8 developed rejection against WT cells (▲) injected twice after 2 months.

B. Ag104 cells were implanted at 3 × 10 ⁵ / mouse in mice immunized against K1735-1D8 and inoculated twice with rejection (■) and control mice injected with PBS (○).

C. Pigmentation of the dorsal skin of mice immunized against K1735-1D8 and rejected against K1735-WT cells.

17. Establishment of established K1735-WT tumors growing in subcutaneous or lung, performed using subcutaneously transplanted K1735-1D8 cells as a vaccine. 17 shows the therapy of established K1735-WT tumors using K1735-1D8 as an immunogen.

A. The mice were vaccinated by subcutaneous injection of K1735-1D8 cells (○) or irradiated K1735-WT cells (□) on the opposite side to mice implanted with K1735-WT tumors having a surface area of 30 mm ² 6 days ago, Mice injected subcutaneously with PBS (■) were included as controls. The same treatment was repeated at the indicated (↓) time point.

B. Untreated mice (left panel) or mice primed with K1735-1D8 8 days after WT cell reception (middle panel) or mice injected with MAb 1D8 primary 8 days after WT cell reception (right panel) Immunohistochemistry of tumors harvested 20 days after transplantation of K1735-WT cells. Each of the upper and lower photographic regions shows CD4 + T cells and CD8 + T cells of the same tumor sinter.

C. Lung metastasis in mice injected intravenously with K1735-WT cells. The top panel shows the lungs of control mice, and the bottom panel shows the lungs of mice vaccinated by repeat transplantation of K1735-1D8 cells.

Splenocytes from mice immunized with K1735-1D8 proliferate when combined with K1735-WT cells, but do not proliferate when combined with cells from sarcoma Ag104 with different antigenicity. 18 shows the proliferation of splenocytes isolated from mice immunized with K1735-1D8.

A. Spleen cells from mice immunized twice with K1735-1D8 or irradiated K1735-WT cells were co-cultured with irradiated K1735-WT ("K1735") or Ag104 ("Ag104") cells for 3 days. . Splenocytes from natural mice were included as controls. Proliferation of tritium-labeled spleen cells was measured.

B. Flow cytometry analysis of proliferation of CD4 + and CD8 + spleen cells labeled with CFDA SE.

19. IFN γ secretion and CTL activity of spleen cells from mice immunized against K1735-1D8. 19 shows IFN-γ secretion and cytotoxic activity.

A. Direct IFN-γ ELISPOT assay on spleen cells from natural mice immunized twice at 10 day intervals using K1735-1D8 cells. Splenocytes harvested 10 days after the last immunization were added to IFN-γ ELISPOT plates and incubated for 24 hours. Splenocytes from natural mice and mice bearing K1735-WT tumors were tested for comparison.

B. In vitro stimulated splenocytes (■) and unstimulated spleen cells (□) from the experiments summarized in Table 1 were tested for IFN-γ secretion by ELISPOT assay performed 30 days after K1735-WT injection. . The average number of spots per group (three) was given for a given mouse (top to bottom) as follows: K1735-1D8 cells were injected 1 day before or 4 or 8 days after WT cell injection; Inject MAb 1D8 1 day before or 4 or 8 days after WT cell injection. The bottom two columns are ELISPOT data for splenocytes from the control group and splenocytes from natural mice.

C. Splenocytes from mice immunized with K1735-1D8 were co-cultured with irradiated K1735-WT cells for 5 days and lysed K1735-WT (■), Ag104 (□) and YAC-1 (○) cells Were tested in a 4 hour Cr ⁵¹ release assay.

D. Experiments in a manner similar to that in FIG. 5C, except that splenocytes were incubated with GM1 antibody and rabbit complement with anti-assial to remove NK cells prior to testing in culture with irradiated K1735-WT cells. Was done. Cytolytic activity against these K1735-WT (■) cells was inhibited by anti-MHC class I Mab (▲). Low cytolytic activity was detected for Ag104 (□), but YAC-1 (○) cells were not lysed.

20. K1735-1D8 cells expressing anti-4-1BB scFv on the surface inhibited tumor formation from mixed K1735-WT cells when the ratio of 1D8: WT cells was 1:10, resulting in "bystander effect". Prove it. 20 shows that a mixture of K1735-1D8 cells and K1735-WT cells (ratio of 1:10) is inhibited from overgrowing in immunocompetent syngeneic (C3H) mice. K1735-WT cells were immunized alone, or K1735-WT cells were mixed so that the ratio of K1735-1D8 transfected cells and untransfected tumor cells: transfected tumor cells was 10: 1. 2 × 10 ⁶ K1735-WT cells were mixed with 2 × 10 ⁵ K1735-1D8 cells and the mixed cells were used to subcutaneously immunize C3H mice. Tumor growth was monitored at 5 day intervals.

21. Sequence of 5B9 anti-human 4-1BB scFv. 21 shows the predicted nucleotide and amino acid sequences of SB9Ig expressed at the cell surface.

Figure 1. Table 1.

Table 2. Table 2.

Table 3. Table 3.

Table 4. Table 4.

Unless otherwise indicated, the practice of the present invention may employ various techniques of molecular biology (including recombination techniques), microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology that belong to the art. Several useful techniques are described, for example, in [MOLECULAR CLONING: A LABORATORY MANUAL, second edition (Sambrook et al., 1989)] and [MOLECULAR CLONING: A LABORATORY MANUAL, third edition (Sambrook and Russel, 2001)] Referred to herein as "Sambrook", [OLIGONUCLEOTIDE SYNTHESIS (MJ Gait, ed., 1984)], ANIMAL CELL CULTURE (RI Freshney, ed., 1987), HANDBOOK OF Experimental IMMUNOLOGY (DM Weir & CC Blackwell, eds.)], [GENE TRANSFER VECTORS FOR MAMMALIAN CELLS (JM Miller & MP Calos, eds., 1987)], [CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., eds., 1987, 2001) [PCR: THE POLYMERASE CHAIN REACTION, (Mullis et al., Eds., 1994)], [CURRENT PROTOCOLS IN IMMUNOLOGY (JE Coligan et al., Eds., 1991)], [THE IMMUNOASSAY HANDBOOK (D. Wild, ed., Stockton Press NY, 1994), BIOCONJUGATE TECHNIQUES (Greg T. Hermanson, ed., Academic Press, 1996), METHODS OF IMMUNOLOGICAL ANALYSIS (R. Masseyeff, W. H.). Albert, and NA Staines, eds., Weinheim: VCH Verlags gesellschaft mbH, 1993), Harlow and Lane (1988) ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publications, New York, and Harlow and Lane (1999). USING ANTIBODIES: A LABORATORY MANUAL Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (collectively also referred to herein as "Harlow and Lane"), Beautage et al. eds., CURRENT PROTOCOLS IN NUCLEIC ACID CHEMISTRY John Wiley & Sons, Inc., New York, 2000)] and Agrawal, ed. Explained in the literature.

The present invention relates to the treatment, prevention and / or alleviation of diseases, disorders and conditions for which an anti-tumor agent or anticancer agent will be beneficial and agents for use therefor.

The present invention provides a variety of methods and compositions useful for generating anti-tumor immunity, for example.

In one aspect, the present invention is directed to the coculture of tumor cells PBMC and PBMC from cancer patients (generally immunocompromised patients), including patients with advanced cancer, to CD3 and CD28 alone or to immobilized antibodies to CD28 and CD40. Novel methods are provided for in vitro obtaining tumor-reactive T lymphocyte populations for in vivo healing use by stimulation. In another aspect, for example, the present invention provides a composition comprising a gene or other polynucleotide encoding an anti-CD3 scFv or anti-4-1BB scFv expressed on the cell surface. In another aspect, the invention provides transfected cells expressing these genes for anti-tumor immunity induction.

Stimulation and activation of T cells using antibodies against CD3 and CD28 immobilized on magnetic beads result in the growth of polyclonal T cells and the production of various cytokines (Levine et al., J Immunol, 159, 5921-30, 1997], Garlie et al., J Immunother, 22, 336-45, 1999). However, there has been no description of the transition from polyclonal proliferation to the generation and / or the proliferation of polyclonal proliferation after stimulation with antibodies to CD3 and CD28.

This can be accomplished, for example, by adding autologous tumor cells to the culture, preferably the initial culture, as described herein. Tumor cells were shown to be destroyed during the same period by monocytes in culture, which were destroyed by activated T cells within 48 to 72 hours and matured into CD83 + dendritic cells. This is believed to be due to exposure to lymphokines such as IFN-γ and TNF-α secreted by activated T cells. Dendritic cells receive killed tumor cells and present tumor antigens to activated T cells, thereby promoting tumor specific T cells to continue to proliferate and overgrow.

In another aspect, the invention provides a gene or other polynucleotide encoding an anti-CD3 single chain antibody fragment (scFv) that may be specific for CD3. Such genes or polynucleotides may be transfected to, for example, expressed on the cell surface of a human or mouse tumor cell line. In both cases, these transfected cells have been shown to activate T lymphocytes, causing them to proliferate to form lymphokines and kill tumor cells.

Further experiments performed in vivo have shown that mouse tumor cells expressing anti-mouse CD3 on the surface induce systemic immunity that can be rejected by immunocompetent mice and reject wild-type cells from the same tumor. Thus, if the scFv encoded by the anti-mouse or anti-human CD3 gene is expressed on the tumor cell surface, it induces polyclonal T cell activation, but the present invention is directed to the polyclonal of anti-CD3 in the presence of tumor cells. It is demonstrated that the activating properties induce metastasis to antigen-specific immunity when administered in vivo as a cancer vaccine.

In another aspect of the invention, for example, an anti-4-1BB single chain antibody fragment (scFv) that can be specific for 4-1BB and can be transfected and expressed on tumor cell surfaces, for example from humans and mice. Construct a gene or other polynucleotide that encodes. Such transfected tumor cells can activate T lymphocytes from each species. Mouse tumor cells transfected with the anti-mouse 4-1BB scFv gene are rejected by immunocompetent mice and can be used as vaccines to induce tumor specific immunity against wild type cells from the same tumor. The immune response has been shown to be memory and may be antigen specific, which is therapeutically effective against studied tumor cells (K1735 melanoma) that grow subcutaneously or grow as lung metastases. These results are biologically significant because K1735 has very low immunogenicity, expresses very low levels of MHC class I molecules, and lacks MHC class II, and is similar for the majority of human tumors.

We have produced genes encoding scFv molecules that are reactive with mouse and human CD3 and 4-1BB. Each gene contains the transmembrane domain and cytoplasmic tail of human CD80. In addition, each gene encodes the hinge, CH2 and CH3 domains of human IgG1 located between the scFv binding site and the transmembrane domain. These genes and cells transfected with these genes are useful for cancer therapy.

As shown, for example, in the following experiments, cDNAs encoding anti-CD3 or anti-4-1BB scFv molecules expressed on the cell surface can be delivered to cancer patients as DNA plasmids or delivered as vectors such as viral or bacterial vectors, etc. have. CDNAs encoding anti-CD3 scFv or anti-4-1BB expressed on the cell surface can be introduced into cancer cells in vitro and gene transduced cancer cells can be used for therapy. Autologous or allogeneic tumor cells can be used. Combinations of scFv genes encoding molecules expressed at the cell surface are included in the present invention, wherein the combination is selected from scFv genes encoding scFv molecules that bind to receptors on T cells that provide activation or costimulatory signals. The invention includes additional methods for in vivo delivery of polyclonal activation signals to T cells, including injecting a patient with a ligand for a surface receptor expressed by the sustained release polymer or T cell containing the antibody. .

The present invention also provides for the in vitro production / proliferation of tumor-selective T lymphocytes to be used for, for example, adoptive delivery to cancer patients by activating tumor-selective T lymphocytes in the presence of autologous tumor cells and signals through CD3 and costimulatory molecules. It provides a new method to make. The present invention facilitates the in vitro production of dendritic cells capable of presenting antigens released from tumor cells to propagate existing tumor-reactive T cell populations, and allows for the generation of immune responses to antigens that were not previously recognized. To facilitate. As described in the present invention, T cells produced in vitro are weakly susceptible to inhibition by TGF-β and have a long lifespan. In addition, the present invention describes two novel types of human tumor vaccines based on the transfection of the scFv gene encoding antibody-derived molecules that recognize CD3 or 4-1BB, which vaccines are highly immunogenic. It demonstrates that it can induce a tumor-destructive immune response when tested for mouse melanoma that is low and expresses very low levels of MHC class I and does not express MHC class II. The approach described herein can be applied to transfection of human tumor cells to express anti-human CD3 or anti-human 4-1BB scFv for use as cell-based vaccines. In addition, it can be readily applied to produce gene-based tumor vaccines that combine genes encoding tumor epitopes with genes encoding anti-CD3 scFv or anti-4-1BB scFv or both. The present invention can be extended to a combination of genes encoding scFv that recognize additional or different immunostimulatory receptors and / or lymphokines that upregulate anti-tumor immune responses.

Although specific experiments will be mentioned and further discussion of the present invention, this is included only as an example and should not be construed as limiting the invention in any way in any situation.

Example 1

CD3-mediated activation of tumor-reactive lymphocytes isolated from human patients with advanced cancer

Peripheral blood mononuclear cells (PBMC) or tumor infiltrating lymphocytes (TILs) were co-cultured with autologous tumor cells in combination with MAb for CD28 or MAb for both CD28 and CD40 in the presence of magnetic beads conjugated with MAb for CD3. Occurring in culture, including destruction of cultured tumor cells, proliferation of lymphocytes and lymphokine production, production of CD83 + APCs and activation / proliferation of tumor-reactive CTLs, as well as decreased lymphocyte susceptibility to inhibition by TGF-β. Some phenomena have been identified.

Patients : Tumors were obtained at the time of surgery of patients with stage IV carcinoma or from malignant exudate (generally ascites) thereof. Tumor and peripheral blood samples were provided with the patient's consent. Most studies were performed on eight patients, five of whom (1OV, 3OV, 8OV, 44OV, 48OV) were ovarian carcinoma, two (1C, 22C) colon carcinoma, and one (1HN) head and neck. Was a carcinoma patient. Cells from 4007, an ovarian carcinoma cell line, were also used.

Preparation of Tumors and Blood Samples : Solid tumors were suspended in media and cells were resuspended after removing fluid from the exudate. Erythrocytes were removed with Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden) and tumor cells were TILed using a Percoll gradient (Sigma, St. Louis, MO). Separated from. Lymphocyte samples were used immediately or stored in liquid nitrogen for later use. According to standard procedures, tumor samples were transplanted in vitro to establish cell cultures. PBMCs containing T lymphocytes, monocytes and B cells were purified using Ficoll-Highpark. Some initial experiments used CD8 + T lymphocytes (greater than 90% pure) selected from TIL using VarioMac magnetic beads (Miltenyi Biotech Inc., Auburn, CA).

Preparation of Culture Combining Lymphocytes, Antibody-Conjugated Beads and Tumor Cells : In an initial experiment, after adding five lymphocytes per tumor cell, the mixture was costar (3513) 12-well plate (Corning, NY, USA). Corning Inc., at 37 ° C with RPMI medium (Gibco, Rockville, Md.) And 10% calf fetal serum (Atlanta Biological, Norcross, GA) Incubated. Then CD3, according to known techniques (Levine, Bernstein et al., J Immunol, 159, 5921-30, 1997), Garlie, LeFever et al., J Immunother, 22, 336-45, 1999). Incubating PBMCs or TILs with or without autologous tumor cells in the presence of magnetic beads conjugated to MAbs for CD28 and / or CD40 (Dynal Inc., Lake Success, NY) The experiment was performed. At this time, beads not conjugated to MAb (or beads conjugated to irrelevant MAb) were used as controls. MAbs are described in 64.1 [Martin, Ledbetter et al., J Immunol, 136, 3282-7, 1986], 9.3 [Martin, Ledbetter et al., J Immunol, 136, 3282-7, 1986] and G28-5 [Ledbetter et al., J Immunol, 138, 788-94, 1987], each of which polyclonally stimulated (anti-CD3), co-stimulated (anti-CD28), or activated APC (anti-CD40). ). When autologous tumor cells were used, the cells (40,000-75,000 / well) were first attached by incubating overnight in Costa 24-well plates containing 2 ml IMDM medium and 10% fetal bovine serum. Then MAb-conjugated beads (3 × 10 ⁶ cells / ml) were added followed by lymphocytes (10 ⁶ cells / ml) in RPMI containing 10% fetal bovine serum. Plates were incubated for 4-5 days in an atmosphere at 37 ° C. containing 6% CO ₂ . The beads were then removed using a magnet, lymphocytes were added to a new well containing medium containing 10 U / ml of IL-2 (Roche Molecular Biochemicals, Indianapolis, Indiana) , Transfer to flask when concentration reached 2x10 ⁶ cells / ml. Cultures were observed to obtain evidence of tumor cell destruction. Cells were counted to measure proliferation of lymphocytes. The medium was sampled to measure TNF production by biological assay using WEHI cells [Espevik and Nissen-Meyer, J Immunol Methods, 95, 99-105, 1986], respectively, and ELISA (EH-IFNG, Woven Endogen, U., Mass.). ), The yield of IFN-γ was measured. TGF-β1 was purchased from Sigma (St. Louis, MO). In all experiments with TGF-β1, this molecule also remained in the culture after removing the MAb-conjugated beads.

CTL analysis . Classical 4-hour ⁵¹ Cr release assay was performed. To characterize effector cells, 10 μg / ml of MAb w6 / 32 (Research Diagnostics Inc., Flanders, NJ) that recognizes MHC class I framework determinants was added Experiments were conducted to inhibit cytotoxicity. In addition, MAb (Beckman Coulter, Brea, CA), anti-CD8 MAb HIT8a (BD Pharmingen, Lexington, KY) and anti-integrin-β2 (CD18) for NK markers CD16 and CD56 ) MAb 60.3 (Beatty et al., J Immunol, 131, 2913-8, 1983) was also used.

FACS analysis of lymphocytes : PE-labeled MAbs were used to assess CD expression density by FACS (Colter Epics XL, Miami, FL), where cells with a predetermined minimum luminance were counted as positive. . To investigate whether the increase in density of CD3 expression that occurred after in vitro activation of lymphocytes was due to selective proliferation of cells that originally had high CD3 expression, PBLs collected from cancer patients were treated with dye CFDA [den Haan et al., J. Exp. Med., 192, 1685-1695, 2000 (Molecular Probes, Eugene, Oregon). This was then incubated in the presence of anti-CD3 / CD28 / CD40 beads for 5 days before the beads were removed and the lymphocytes were grown in medium containing 10 U / ml IL-2. FACS analysis was performed at two time points (times 4 and 3) after bead removal, and cells were analyzed for CFDA brightness and CD3 expression. Labeled lymphocytes incubated with control beads were studied for comparison.

Demonstration of low levels of T cell reactivity in the absence of stimulation through antibody-conjugated beads : 6 initial stages, after incubating CD8 + T lymphocytes purified from TIL with tumor cells, supernatants analyzed for TNF or IFN-γ The experiment was conducted. In a representative experiment, CD8 + TILs from colon cancer patients (1C), first cultured with 1C tumor cells, were isolated and added to either fresh 1C cells or tumor cells from lung carcinoma patients (3L). Small amounts of TNF (1.2 pg / ml) were detected when 1C lymphocytes were combined with 1C, but not when combined with 3L tumors, and TNF and IFN-γ when TILs from 3L were combined with 3L tumor cells. (1.5 pg / ml) was produced but not in culture alone. There was no evidence of lymphocyte proliferation. In subsequent experiments, a TIL population comprising CD8 + lymphocytes in addition to monocytes, CD4 + T cells and B cells was combined with autologous tumor cells and cultured for 10-15 days. Approximately 10-fold higher levels of TNF (4.5-48 pg / ml) and IFN-γ (up to 150 pg / ml) were detected in supernatants obtained from cultures of 8 of 13 patients. There was still no lymphocyte proliferation.

Demonstration of T Cell Proliferation and Tumor Cell Destruction in the Presence of Autologous Tumor Cells and Anti-CD3- Conjugated Beads : After initial experiments, experiments are performed in systems that induce signals through various lymphocyte receptors using MAb-conjugated magnetic beads. (Levine, Bernstein et al., J Immunol, 159, 5921-30, 1997), Garlie, LeFever et al., J Immunother, 22, 336-45, 1999). In the presence of beads conjugated to MAb for CD3 and MAb for CD28, PBMC or TIL, alone or in combination with CD40, were combined with autologous tumor cells. Similar groups were included, including lymphocytes but no tumor cells. As a control, lymphocytes were incubated with the control non-conjugated beads with or without tumor cells. After 3-5 days the beads were removed and lymphocytes and tumor cells were incubated separately for 10-21 days with 10 U / ml IL-2.

1 shows the results of an experiment that TILs actively proliferate from patient OV44 when exposed to anti-CD3 / CD28 / CD40 conjugated beads for 4 days. Lymphocytes cultured in the absence of CD3 signal did not proliferate, nor did lymphocytes cultured with anti-CD28 and / or CD40 beads proliferate (data not shown). Autologous tumor cells proliferated more if they were present from the beginning with beads that induce signaling through CD3 (Panel B). Anti-CD3 / CD28 conjugated beads induced proliferation similarly to those with anti-CD3 / CD28 / CD40 conjugated beads (data not shown).

2 shows the results of experiments in which PBLs from patients 1C and various MAb-conjugated beads were incubated with autologous tumor cells or allogeneic cells for 5 days. Similar to the results illustrated in FIG. 1, the number of lymphocytes per culture was higher than that of CD3 / CD28 (Panel C) or anti-CD3 / CD28 / CD40 (Panel D) activated lymphocytes combined with 4007 cells. It was much higher in combination with 1C tumors. FACS analysis revealed that more than 90% of the activated lymphocytes expressed CD3, of which approximately 70% were CD8 + and less than 5% expressed either CD16 or CD56. On the other hand, if the beads did not provide a signal through CD3 (Panel A and Panel B), they were more proliferated with the addition of allogeneic cells, presumably an immune response to homologous antigens expressed on 4007 cells. something to do.

Most tumor cells are destroyed within 24 to 48 hours after exposure to autologous lymphocytes in the presence of anti-CD3 / CD28 or anti-CD3 / CD28 / CD40 conjugated beads, often causing the culture to contain only completely lymphocyte-shaped cells. . To study whether such tumor destruction has immunological specificity, a series of PBL dilutions (10 ⁶ to 10 ⁵ / sample) from cancer patients are combined with tumor cells or fibroblasts from autologous tumor cells or allogeneic donors Four experiments were performed. In both experiments, there was approximately 10-fold more TNF in the culture supernatant in the presence of autologous tumors, but there was no difference in the killing of cells from autologous or allogeneic tumors or allogeneic fibroblasts. It was concluded that tumor cell destruction that appeared after 24 to 72 hours in the presence of lymphocyte activation was not antigen specific, probably because a large amount of activated T lymphocytes and lymphokines blocked any specific component.

Generation of Tumor-Selective CTLs : MHC-class I-limited CTLs were generated from tumor cells and lymphocytes activated by anti-CD3 / CD28 or anti-CD3 / CD28 / CD40 beads. FIG. 3 shows the results of experiments with PBLs from patient 1C, which were activated in the experiment shown in FIG. 2. After activation with tumor cells and MAb-conjugated beads, beads were removed and lymphocytes were propagated in 10 U / ml IL-2 medium over 3 weeks without adding tumor cells and beads. PBLs activated by 1C and anti-CD3 / CD28 beads showed potent cytolysis against 1C cells, and lysis was inhibited by MAb and anti-MHC class I framework MAb w6 / 32 for CD8 (FIG. 3A). Allogeneic 4007 cells only killed 20% when E / T = 50: 1, compared to 98% lysis of 1C cells (FIG. 3A). 3B provides similar data for PBL stimulated with anti-CD3 / CD28 / CD40 beads. 4007 cell lysis at this time was at the same low level as the 1C case in the presence of MAb w6 / 32. In contrast, PBL stimulated with anti-CD3 / CD28 / CD40 beads killed both 1C and 4007 cells even in the presence of MAb or MAb w6 / 32 for CD8 (data not shown). CD8 + cells from the cell population used in the experiments shown in FIG. 3B lysed 25% 1C cells at an E / T ratio of 20/1, but 0% of cells in 4007 cell lines and 0% of cells in allogeneic B cell lines. Dissolved. The lysis rate of 1C cells in this experiment was 5% in the presence of MAb w6 / 32 and 5% in the presence of anti-CD18 MAb 60.3, only 25% when using a combination of MAb for CD16 and MAb for CD56. At 18%. Lymphocytes activated by coculture with 4007 cells and any beads did not selectively lyse 1C or 4007 cells. Similar results were obtained by repeating the CTL analysis twice.

Production of Th1-type lymphokine : A large amount of IFN-γ was detected in culture supernatants from lymphocytes activated through CD3. This is illustrated in FIG. 4, which also indicates that IFN-γ is produced more when autologous tumor cells are present on the first 4-5 days of culture.

Table 1 shows six additional representative experiments demonstrating their proliferation and lymphokine production when tested immediately after PBL or TIL harvested from a patient or after in vitro activation with beads. Anti-CD3, anti-CD3 / CD28, anti-CD3 / CD40 and anti-CD3 / CD28 / CD40 beads significantly increased lymphocyte proliferation with no significant difference between them. In contrast, anti-CD28, anti-CD40 and anti-CD28 / CD40 beads alone did not increase lymphocyte proliferation and lymphokine production compared to control beads, indicating that signaling through CD3 is important. The production of TNF and IFN-γ were correlated. This amount decreased to background levels when lymphocytes were grown without tumor cells and beads for more than 3-5 days. 1 and 4, CD3 signaling was required to induce vigorous lymphocyte proliferation and lymphokine production.

Upregulation of CD3 and other markers on activated lymphocytes via MAb-conjugated beads : additionally without beads before and after incubation of lymphocytes with tumor cells and anti-CD3 / CD28 / CD40 beads for 3-5 days CD antigen expression density in the lymphocyte population was measured by FACS after 3-7 days propagation. To reflect the change in density of CD receptor expression, the percentage of cells in which the luminance of cells in each population was equal to or higher than the pre-selected density was reported (Table 2); Unstimulated PBLs from six healthy donors (ages 30-65) were analyzed for comparison. Unstimulated PBLs from cancer patients had low levels of CD3, CD4 and CD28. In addition, CD8 density was lower in 4 of 5 patients, but CD86 density was higher than in unstimulated PBLs from healthy donors. When PBL was incubated with control beads, some CD3 expression was increased, but CD28 expression was not significantly increased. In contrast, incubation with anti-CD3 / CD28 / CD40 beads consistently restored CD3 and CD28 expression and doubled the number of cells expressing CD8 at high density. CD3 expression density was studied in TILs from five patients. The results were 2.9%, 40.2%, 96%, 42.8% and 40.1%, respectively, ie larger deviations and generally higher than for PBMCs. CD8 expression by TIL was higher than in the case of PBMC and increased from 61.4% to 87.3%. Corresponding values for CD28 expression in TIL were 39.3% and 52.8%.

To investigate whether the increase in density of CD3 expression following in vitro activation of lymphocytes is due to the selective proliferation of cells that originally had high CD3 expression, PBLs collected from cancer patients were treated with the dye CFDA (Molecular Probe, Eugene, Oregon, USA). Was labeled). Experiments were performed in which 40.2% originally labeled CD3 expressing CD3 with the dye CFDA [den Haan, Lehar et al., J. Exp. Med., 192, 1685-1695, 2000]. CD3 expression increased to 95% after activation via anti-CD3 / CD28 / CD40 beads. FACS analysis using CFDA and PE-labeled anti-CD3 as probes revealed no selective proliferation of PBL subpopulations which originally had higher CD3 expression.

CD83 expression in culture after stimulation with anti-CD3 / CD28 beads: Stimulation of PBMCs (10-20% of which express monocyte marker CD14) by anti-CD3 / CD28 / CD40 beads resulted in dendritic cells In order to investigate whether the maturation of the cells can be increased, CD83 expression levels were measured at various time points after stimulation. Mature dendritic cells expressed CD83 but did not express immature dendritic cells or blood monocytes. 5, which illustrates a typical experiment, shows that CD83 expression was detected in 35% of cells even after only 24 hours of stimulating PBMC with anti-CD3 / CD28 beads. CD83 expression was not detected in PBMC (prior to stimulation) on day 0.

To determine which cells express CD83 after PBMC stimulation, fluorescein labeled anti-CD3 and PE- on day 2 after stimulation with anti-CD3 / CD28 beads or anti-CD3 / CD28 / CD40 beads Two-color staining was performed with labeled anti-CD83. FIG. 6 shows that CD83 is not expressed on cells in the absence of bead activation, whereas beads conjugated with anti-CD3 / CD28 induced CD83 expression on a distinct population of CD3 negative cells (13.9%) and also had a significant proportion of CD3. CD83 expression was also induced on positive cells. In contrast, the beads conjugated with anti-CD3 / CD28 / CD40 induced CD83 expression, but at lower levels than with anti-CD3 / CD28 beads in both CD3 negative and CD3 positive cells. This result indicates that cells expressing the dendritic cell marker CD83 rapidly induced from PBMC after stimulation with beads conjugated with anti-CD3 and anti-CD28 MAbs. Stimulation by beads conjugated with anti-CD3, anti-CD28 and anti-CD40 MAbs in stimulation of CD83 expression was not as effective as stimulation by beads conjugated with anti-CD3 and anti-CD28 MAbs.

Increasing resistance to inhibition by TGF-β1 in the presence of activation signals through MAb-conjugated beads : Table 3 shows that the inhibitory effect of TGF-β1 on lymphokine production and lymphocyte proliferation induces signals through CD3. Five representative experiments were conducted to investigate whether they could be altered by coculture with the beads. For control beads, the levels of TNF and IFN-γ were low, which was further inhibited by TGF-β1. In contrast, in the case of anti-CD3 / CD28 / CD40 beads, this level increased to a level close to that seen in the absence of TGF-β1. Likewise, the inhibitory effect of TGF-β1 on lymphocyte proliferation was much less with anti-CD3 / CD28 / CD40 beads and not at all with patient 1HN. Relative resistance to T cell proliferation and lymphokine production also occurred when the dose of TGF-β1 was increased to 20 ng / ml and the lymphocyte concentration was reduced to 10 ⁵ / sample (data not shown). . Beads stimulating with CD28 or CD40 alone or through both did not protect against TGF-β1 (data not shown).

Thus, lymphocyte activation in the presence of tumor cells involves the death of tumor cells and results in the release of antigen. Monocytes in culture receive tumor antigens, differentiate into CD83 positive APCs, and present epitopes for selective proliferation of tumor-reactive T cells. Therapeutic vaccines can activate and proliferate inhibited lymphocytes in individuals with tumors according to the same principle, and can also facilitate the generation of immune responses against subdominant epitopes. In general, the culture system for the production of tumor-reactive T cells will comprise four components. These are:

1) T cells from cancer patients,

2) antigen presenting cells from the patient,

3) beads conjugated with anti-CD3 and anti-CD28 antibodies or beads conjugated with anti-CD3, anti-CD28 and anti-CD40 antibodies,

4) tumor cells from the patient.

Various modifications will be devised or appreciated for this component. This includes modifications to the source of the components as well as modifications to the timing of addition of any of the four components. For example, a patient's T cells may be isolated from peripheral blood or from tumor infiltrating lymphocytes. The antigen presenting cells shown in the examples were present in peripheral blood mononuclear cell fractions, but may be derived from other sources, such as, for example, bone marrow. In the examples, autologous tumor cells were used in culture, but since allogeneic antigens are presented by autologous APCs, allogeneic tumor cells or tumor antigens may be used in place of or in addition to autologous tumor cells. Magnetic beads conjugated with anti-CD3 and anti-CD28 antibodies can be replaced with antibodies immobilized in other ways and added to additional cell surface receptors that promote activation of polyclonal T cells and proliferation of tumor-reactive T cells. It may also consist of specific immobilized antibodies or ligands.

In addition, the procedures used can produce CD3-positive lymphocytes, allowing them to continue to proliferate in culture in excess of 10 weeks and are useful for adoptive immunotherapy. This is because co-stimulation through CD28 reduces the likelihood of apoptosis in lymphocytes (Boise et al., Immunity, 3, 87-98, 1995), Daniel et al., J Immunol, 159, 3808- 15, 1997]) may extend prolonged in vitro life [Levine, Bernstein et al., J Immunol, 159, 5921-30, 1997]. Costimulated lymphocytes also survived for a long time after returning to autologous patients as opposed to lymphocytes proliferated in the presence of high doses of IL-2 [Ranga et al., Proc. Natl. Acad. Sci., 95, 1201-1206, 1998]. T cell stimulation via CD3 in combination with CD28 alone or CD3 in combination with CD28 and CD40 was associated with lymphocyte proliferation and TNF and IFN-γ, even when TGF-β1 was used at a saturation level of 20 ng / ml in culture. It should be noted that approximately 50% protection of TGF-β1-mediated inhibitory effects on production can be achieved.

Example 2

Construction, transfection and expression of anti-CD3 scFv at the surface of a vector encoding anti-human and anti-mouse CD3 scFv of human or mouse origin induce polyclonal stimulation to proliferate T cells and to form a Th1 rim Demonstration of the ability to produce focaine, to be cytolytic, and to have anti-tumor activity in vivo

The experiments described above indicate that the signals provided by anti-CD3 mAbs conjugated to magnetic beads activate and proliferate tumor-reactive lymphocytes in cultures containing autologous tumor cells and PBMCs or TILs. The gene was a gene designed for tumor therapy that expresses anti-CD3 mAb single chain Fv (scFv) derivatives on tumor cell surfaces. Anti-CD3 scFv responsive to mouse CD3 was constructed from hybridoma 500A2 and anti-CD3 responsive to human CD3 were constructed from hybridoma G19-4 [Ledbetter et al., J. Immunol., 136, 3945 -3952, 1986].

Production of scFv: to clone the light chain and heavy chain variable domains of an antibody from a hybridoma RNA was produced in the cell surface in the form of single chain Fv (scFv) ([Hayden et al, Ther Immunol, 1, 3-15, 1994.] , Gilliland et al., Tissue Antigens, 47, 1-20, 1996). RPMI containing hybridomas [10% fetal bovine serum, 4 mM glutamine, 1 mM sodium pyruvate and 50 u / ml penicillin-streptomycin (both Life Technologies, Gaitsburg, MD, USA)] Cells were harvested after growing in medium and maintaining logarithmic growth for several days. Cells were harvested by centrifugation of the suspension culture, and either Trizol or QIAGEN RNA columns (Life Technologies, Reuters, MD, and Qiagen, Valencia, Calif.) Were directed to the manufacturer. RNA from 2 × 10 ⁷ cells was isolated either by use or by modifying the NP-40 lysis technique (Gilliland, Norris et al., Tissue Antigens, 47, 1-20, 1996). Using 1 μg of total RNA and using a Superscript II reverse transcriptase (Life Technologies) and a random hexamer (Takara Shuzo, Otsu Shiga, Japan), a randomly primed first strand of cDNA was used. Synthesized. After reverse transcription, poly G-tails were added to cDNA fragments using dGTP and end group transferase (an enzyme that catalyzes the addition of deoxyribonucleotides from deoxynucleotide triphosphate to the terminal 3'-OH group of the DNA strand). made. An anchor tail was made using an unknown leader peptide at one end of the cDNA to increase mRNA cloning efficiency. The 5 'primer contains a poly C tail as described for PCR of T cell receptor chain sequences (Loh et al., Science, 243, 217-20, 1989), but with SacI, XbaI and EcoRI sites for cloning. It was a modified ANCTAIL primer. The sequence was as follows:

Primers for the cDNA 3 'end were obtained in the heavy or light chain constant region and bound approximately 50 bases above the J-C junction. Each 3 'primer contained a HindIII, BamHI and Sal I site for cloning. Thus, restriction sites for the initial subcloning fragments were incorporated as part of these original PCR amplification primers, and the amplified PCR fragments were digested and placed into pUC19, pSL1180 or TOPO vectors (Invitrogen, San Diego, Calif.). Subcloning was sequenced. DNA sequencing was performed using pUC, T7 or M13 pluripotent primers and reverse primers and BigDye Terminator Cycle sequencing kit reagents in ABI Prism 310 (PE Biosystems) sequencer. This was performed on miniprep DNA (Kiagen, Valencia, CA) using Foster Biosystems, Foster City.

Once the individual variable domains were isolated and consensus sequences were generated from three or more identical clones, scFvs were constructed by PCR amplification using overlapping oligonucleotides that fused cDNAs encoding light and heavy chain variable regions. . During this sealing PCR, ₃ linkers (gly ₄ ser) were added as part of the overlapping oligonucleotides to link the light and heavy chain variable domains [Gilliland, Norris et al., Tissue Antigens, 47, 1-20]. , 1996]. The assembled scFv molecules were subcloned upstream of the human IgG1 hinge, CH2, and CH3 domains in-frame fused to human CD80 transmembrane and cytoplasmic tail (Winberg et al., Immunol Rev, 153, 1996). The completed expression cassette encoded the natural leader peptide for the light chain V region or secretory signal peptide from the L6 VK light chain fused to the light chain variable region of scFv at the SalI site. The scFv was encoded as a HindIII-BclI or SalI-BclI cassette, wherein the first restriction site was encoded within the frame of the open reading frame, while the second restriction site was in the frame of the reading frame for the fusion protein. Was off. This cassette was then fused to the human IgG1 wild type Fc domain encoded on the BglII-BamHI fragment. CD80 transmembrane and cytoplasmic tails from human amygdala RNA were amplified by PCR, which was encoded on BstBI-CIaI fragments containing stop codons immediately upstream of the ClaI site. Each subfragment was subcloned into a synthetic polylinker / multiple cloning site inserted therein by modifying the vector pCDNA3. Once the complete fusion protein construct encoding the cell surface scFv was assembled, the entire expression cassette was used as a HindIII-ClaI fragment to retroviral expression vector pLNCX [Miller and Rosman, Biotechniques, 7, 980-2, 984-6, 989-90]. , 1989] (FIG. 9).

Transfection : Plasmid DNA was prepared from the recombinant retroviral vector, which was then subjected to CaPO ₄ precipitation technique [Winberg, et al., (1996) Immunol Rev. 153: 209-223.] To transfect PT67 dual tropic (Clontech, Palo Alto, CA) packaging cells. In summary, DMEM containing 10% fetal bovine serum, 4 mM glutamine, 2 × DMEM non-essential amino acids, and penicillin-streptomycin (hereinafter referred to as this agent DMEM-C; all reagents are from Life Technologies) Plated at approximately 25% density in vitro and grown overnight before transfection. Plasmid DNA was added to 0.5 ml of 0.25 M CaCl ₂ and then added dropwise to 0.5 ml of 2 × HEBS buffer, pH 7.1. After precipitation for 5 minutes at 37 ° C., the solution was added dropwise to the cells in a 100 mm culture dish containing fresh DMEM-C (8 ml). The transfected cells were incubated overnight then washed twice with PBS and fed fresh medium. Viral supernatants were harvested from the transfected cells and transduced using them after 24 hours. Alternatively, adherent PT67 cells transfected and expressing cell surface scFv were co-cultured with B cell lines grown in suspension. After several passages the packaging cells were diluted from the cultures and cell-surface scFv could be expressed by panning the B cell line with goat anti-human IgG1 immobilized in the culture flask. Cells expressing high levels of cell surface scFv bound more tightly to the flasks, negative cells and low level expressing cells were washed out of the flasks. High level expressing cells were then scraped off from the flask surface and isolated and recultivated for several days before being used for biological analysis.

Mice and Tumor Cell Lines : Female C3H / HeN mice, 6-8 weeks of age, were purchased (Taconic, Germantown, NY). K1735 is a melanoma of C3H / HeN origin with the metastasis clone M2 selected (Fidler and Hart, Cancer Res, 41, 3266-3267, 1981). Consistent with previous reports, its amount of MHC class I expression was very low (data not shown). Animal facilities were ALAC approved and protocols were approved by the appropriate Institutional Animal Committee.

Antibodies : R-Phycoerythrin (PE) -conjugated MAbs GK1.5 (anti-mouse CD4), 53-6.7 (anti-mouse CD8a) and purified AF3-12.1 (anti-H-2K ^K ) (From San Diego, Calif.) And R-PE conjugated Goat F (ab ') ₂ anti-human IgG was purchased from Biosource International (Camarillo, Calif.). MAb 169-4 (anti-CD8) is known. Obtained from Dr. R. Mittler (Emery College, Atlanta, GA). GK1.5 (anti-CD4) was prepared from hybridomas obtained from ATCC.

Transfection of Vectors and K1735 Cells : Variable region cloning, scFv production and generation of scFv expression are described in Gilliland, Norris et al., Tissue Antigens, 47, 1-20, 1996, Hayden et al., Tissue Antigens, 48, 242-54, 1996, Winberg, Grosmaire et al., Immunol Rev, 153, 1996. The study was carried out using variable region genes from anti-CD3 hybridoma 500A2 or anti-4-1BB hybridoma 1D8 for surface expression of cell-bound 500A2 scFv or 1D8 scFv (Hayden, Grosmaire et. al., Tissue Antigens, 48, 242-54, 1996, Winberg, Grosmaire et al., Immunol Rev, 153, 1996). transmembrane domain and cytoplasmic tail from CD80 to express scFv (because it mediates cytoskeletal adhesion and crosslinking during cell-cell contact (Doty and Clark, J Immunol, 157, 3270-9, 1996), [ Doty and Clark, J Immunol, 161, 2700-7, 1998]). The scFv gene fusion construct in pLNCX was transfected into RetroPack ^TM PT67 packaging cells (Clontech laboratories, Inc., Palo Alto, CA) following CaPO ₄ precipitation. Medium from these cells was used to transfect K1735-WT cells. To confirm scFv surface expression, G418 resistant clones were stained with PE labeled goat anti-human IgG.

Animal studies : 2 × 10 ⁶ K1735-WT or K1735-500A2 cells or gamma-irradiated (12,000 rads) K1735-WT cells were subcutaneously transplanted to the dorsal side of mice (5 or 10 mice / group). Immunized mice were injected with K1735-WT (2 × 10 ⁶ cells / mouse) or Ag104 (3 × 10 ⁵ cells / mouse). Tumor size was assessed by measuring the two largest vertical diameters with calipers and reported as mean tumor area (mm ² ) ± SD. The tumors subcutaneously implanted in the mice were shaved to facilitate tumor measurement.

In vivo depletion of CD4 + and / or CD8 + T lymphocytes and NK cells : MAb for CD4 (GK1.5, rats, as described in Chen, Ashe et al., Cell, 71, 1093-102, 1992). T cells were depleted by three intraperitoneal injections of IgG2b) or MAb (169-4, rat IgG 2a) or CD8 to CD8 or 0.5 mg / mouse for three consecutive days. Subsequently, 0.5 mg of each MAb was injected every 3 days to maintain depletion. NK cells were depleted by intraperitoneal injection of anti-Asiaal GM1 antibody at 30 μl / mouse every four days. On day 12, spleen cells from each group were analyzed by FACS to confirm the depletion efficiency. The mice were then implanted subcutaneously with tumor cells.

Proliferation Assay : Spleen cells were seeded (1 × 10 ⁵ cells / well) in 96-well flat bottom plates with irradiated (3,000 rads) 5 × 10 ⁵ syngeneic spleen cells (used as APC) and tumor cells. . After incubation for 72 hours, three cultures were pulsed with 1 μCi ³ [H] thymidine (Amersham Pharmacia, Biotech Piscataway, NJ) for 16-18 hours and the incorporation was measured. It was.

To investigate the proliferation of T cells in vitro, spleen cells were prepared using 2 μM CFDA SE (5- (and-6) -carboxyfluorescein diacetate, according to the manufacturer's protocol (Molecular Probes, Eugene, OR). Incubated with succinimidyl ester) and labeled [den Haan, Lehar et al., J. Exp. Med., 192, 1685-1695, 2000], K1735-WT cells were incubated for 3 days with or without addition and analyzed by FACS.

CTL Activity Assay : Two to four weeks after transplanting K1735-1D8, mice were sacrificed and spleen cell suspensions were prepared. If indicated, NK cells were removed using anti-Asialo GM1 antibody and rabbit complement (Cedarane, Ontario, Canada). 5 × 10 ⁶ spleen cells were incubated for 5 days with 1 × 10 ⁵ K1735-WT cells irradiated (12,000 rads) in 24-well plates (Costar Corp., Cambridge, Mass.). . Cytolytic activity was examined by a 4-hour Cr ⁵¹ release assay at various E / T ratios.

ELISPOT Assay : Murine IFN-γ ELISPOT kit (R & D Systems, Minneapolis, MN) was used according to the manufacturer's protocol, and plates were plate-scanning service (Cell Technology, Cleveland, Ohio). (Cellular Technology Ltd.).

Polyclonal activated human T cells proliferate to produce Th1-type lymphokine and become cytolytic : anti- on the cell surface of Reh (reticulocyte endothelial cell line) and T51 (B cell lymphoblastoid cell line) Human CD3 scFv expression is shown in FIG. 10. The transfected cells were shown to express high levels of anti-CD3 scFv gene product.

Induction of T cell proliferation of transfected cells versus wild type Reh and T51 cells was tested by culturing the cell line with PBMCs from normal donors. Wild type and transfected cell lines were treated with mitomycin C to prevent proliferation during culture. Transfected Reh and T51 cells expressing anti-CD3 scFv induced proliferation in a dose dependent manner, but not for wild type Reh and T51 cells (FIG. 11).

An experiment was conducted to test whether anti-human CD3 scFv expression on tumor cell surface stimulated T cells from PBMCs to rapidly kill transfected cells. To cover the wild type or transfected Reh and T51 cells with ⁵¹ Cr, and using PBMC from healthy donors was performed for 8 hours ⁵¹ Cr release assay. 12 shows that transfected cells were rapidly killed by resting T cells, significantly releasing ⁵¹ Cr in a dose dependent manner, but not wild type cells.

Rejection of immunocompetent syngeneic mice against K1735-500A2 cells : As shown in FIG. 7, K1735-500A2 cells transfected and expressing anti-mouse CD3 scFv are transiently grown in mice and the mice reject the response thereto Cause Cells expressing CD80 grew slowly but progressively than non-transfected cells as previously reported (Chen et al., J Exp Med, 179, 523-532, 1994).

Ten mice were transplanted three times at 7 day intervals with 2 × 10 ⁶ anti-CD3 (500A2 scFv) transfected cells (without tumor). Nine mice as controls were injected with only PBS. Subsequently, 10 ⁶ K1735-wT cells were injected into both groups. K1735-WT cells formed tumors in all control mice, but no tumor developed in 6 of 10 immunized mice. Tumor growth in four tumor-immunized mice was delayed compared to the non-immunized group (control). There was no evidence of toxicity or immunosuppression in any of the mice, including mice injected repeatedly with anti-CD3 scFv-transfected tumor cells.

Demonstration of bystander effect when K1735-500A2 cells are mixed with K1735-WT cells : Tumor cells expressing anti-CD3 scFv in vivo by, for example, transfection in vivo, have an effective immune response against wild-type tumor cells. To investigate whether or not to induce, two experiments were performed in which K1735-WT cells were mixed with K1735-500A2 cells. The first experiment was performed by mixing two types of cells in equal numbers, in which case the tumors regressed after short-term in vivo growth. The second experiment was performed by mixing 2 × 10 ⁶ K1735-WT cells with 2 × 10 ⁵ K1735-500A2 cells. As shown in FIG. 13, the overgrowth of WT cells was inhibited when compared with the transplantation alone.

Immunization with K1735-500A2 Cells Causes Proliferation of Tumor-Selective T Cells : Splenocytes were harvested from mice that rejected the transplanted K1735-500A2 cells. 14 shows that these spleen cells proliferate more in combination with irradiated K1735-WT cells than in combination with irradiated cells from syngeneic sarcoma Ag104 antigenically distantly. Indicates. Splenocytes from mice immunized with irradiated K1735-500A2 cells did not proliferate more than splenocytes from native (control) mice.

Thus, anti-CD3 scFv expression on tumor cell surface induces rapid killing of tumor cells and propagates T cells. These properties promote tumor specific immunity, in which the destruction of tumor cells and polyclonal activation of T cells produce tumor antigens that are accepted by dendritic cells that mature under the influence of cytokines produced by T cells. Because it becomes. T cells first become susceptible by polyclonal anti-CD3 activation and then recognize tumor antigens presented by APC and continue to grow tumor specific T cells. The fact that not only type 1 lymphokines formed by activated T cells but also T cells themselves can destroy bystander tumor cells suggests that transfection of tumor cells expressing anti-CD3 scFv in vivo may be therapeutically effective. Instruct.

Example 3

Anti-4-1BB scFv for Gene Therapy of Cancer

Established techniques for producing vaccines that stimulate the immune system (Hayden, Grosmaire et al., Tissue Antigens, 48, 242-54, 1996, Winberg, Grosmaire et al., Immunol Rev, 153, 1996). Vectors encoding cell-bound single chain Fv fragments from hybridoma 1D8 (anti-4-1BB monoclonal antibody) [Melero, Shuford et al., Nat Med, 3, 682-5, 1997] using Was produced. This vector was derived from cells from K1735 melanoma with low immunogenicity and very low MHC class I expression [Ward et al., J. Exp. Med., 170, 1989]. Transfected cells induce a strong Th1 response, which requires CD4 + T lymphocytes rather than CD8 + T lymphocytes and is associated with NK cells. The vaccinated mice developed rejection against wild type K1735 tumors growing in subcutaneous swelling or lung. The approach of the present invention will be effective against micrometastasis in human patients, including, for example, human patients with low MHC class I expression levels.

Mice and Tumor Cell Lines : Female C3H / HeN mice, 6-8 weeks of age, were purchased (Taconic, Germantown, NY). K1735 is a melanoma of C3H / HeN origin with the metastasis clone M2 selected (Fidler and Hart, Cancer Res, 41, 3266-3267, 1981). Its MHC class I expression amount was very low (data not shown). Ag 104 [Ward, Koeppen et al., J. Exp. Med., 170, 1989] is a spontaneous fibrosarcoma of C3H / HeN. YAC-1 was obtained from the American Type Culture Collection (Rockville, MD). Animal facilities were ALAC approved and protocols were approved by the appropriate laboratory animal control committee.

Antibodies : R-Phycoerythrin (PE) -conjugated MAbs GK1.5 (anti-mouse CD4), 53-6.7 (anti-mouse CD8a) and purified AF3-12.1 (anti-H-2K ^K ) (San Diego, Calif.) And R-PE conjugated Goat F (ab ') ₂ anti-human IgG were purchased from BioSource International (Camarillo, Calif.). MAb 169-4 (anti-CD8) was purchased and GK1.5 (anti-CD4) was prepared from hybridomas obtained from ATCC. Rabbit anti-asialo GM1 antibody was purchased from Wako Pure Chemical Industries (Richmond, VA), and purified rat IgG from Sigma and Rockland (Gilbertsville, PA). Purchased.

Transfection of Vectors and K1735 Cells : Variable region cloning, scFv production and production of scFv expression are described in Gilliland, Norris et al., Tissue Antigens, 47, 1-20, 1996, Hayden, Grosmaire et al. , Tissue Antigens, 48, 242-54, 1996, Winberg, Grosmaire et al., Immunol Rev, 153, 1996. This study surface expresses cell-bound 1D8 scFv (Hayden, Grosmaire et al., Tissue Antigens, 48, 242-54, 1996, Winberg, Grosmaire et al., Immunol Rev, 153, 1996). To a variable region gene from anti-4-1BB hybridoma 1D8 (Melero, Shuford et al., Nat Med, 3, 682-5, 1997). CD80, which mediates cytoskeletal adhesion and crosslinking during cell-cell contact to express scFv (Doty and Clark, J Immunol, 157, 3270-9, 1996), Doty and Clark, J Immunol, 161, 2700-7 , 1998 transmembrane domain and cytoplasmic tail. The scFv gene fusion construct in pLNCX was transfected into RetroPak ^™ PT67 packaging cells (Clontech Laboratories Inc., Palo Alto, CA) according to CaPO ₄ precipitation. Medium from these cells was used to transfect K1735-WT cells. To confirm scFv surface expression, G418 resistant clones were stained with PE labeled goat anti-human IgG.

Animal studies : 2 × 10 ⁶ K1735-WT or K1735-1D8 cells or gamma-irradiated (12,000 rads) K1735-WT cells were subcutaneously transplanted to the dorsal side of mice (5 or 10 mice / group). Immunized mice were injected with K1735-WT (2 × 10 ⁶ cells / mouse) or Ag104 (3 × 10 ⁵ cells / mouse). Mice with established K1735-WT tumors were implanted subcutaneously with K1735-1D8 (2 × 10 ⁶ cells / mouse); Immunized cells were transplanted to the opposite side of the WT cells in the back. Tumor size was assessed by measuring the two largest vertical diameters with calipers and reported as mean tumor area (mm ² ) ± SD. The tumors subcutaneously implanted in the mice were shaved to facilitate tumor measurement.

In one experiment, 3 × 10 ⁵ K1735-WT cells were injected intravenously into the tail lateral vein of a mouse to establish lung metastasis (Kahn et al., J Immunol, 146, 3235-3241, 1991). Three days later, K1735-1D8 cells were implanted subcutaneously on one side of the back and repeated four times at weekly intervals. Mice were sacrificed 37 days after implantation of WT cells. When intratracheal injection of Indian ink (15% in phosphate buffered saline) was taken out of the lungs, the unstained transition was in contrast to normal black tissue [Estin et al., Proc Natl Acad Sci USA, 85, 1052-6, 1988].

In vivo depletion of CD4 + and / or CD8 + T lymphocytes and NK cells : MAb for CD4 (GK1.5, rats, as described in Chen, Ashe et al., Cell, 71, 1093-102, 1992). T cells were depleted by three intraperitoneal injections of IgG2b) or MAb (169-4, rat IgG 2a) or CD8 to CD8 or 0.5 mg / mouse for three consecutive days. Subsequently, 0.5 mg of each MAb was injected every 3 days to maintain depletion. NK cells were depleted by intraperitoneal injection of anti-Asiaal GM1 antibody at 30 μl / mouse every four days. On day 12, spleen cells from each group were analyzed by FACS to confirm the depletion efficiency. The mice were then implanted subcutaneously with tumor cells.

Proliferation assay : Spleen cells were seeded (1 × 10 ⁵ cells / well) in 96-well flat bottom plates with irradiated (3,000 rads) 5 × 10 ⁵ syngeneic spleen cells (used as APC) and tumor cells. . After incubation for 72 hours, three cultures were pulsed with 1 μCi ³ [H] thymidine (Amersham Pharmacia, Biotech Piscataway, NJ) for 16-18 hours and the incorporation was measured.

To investigate the proliferation of T cells in vitro, spleen cells were prepared using 2 μM CFDA SE (5- (and-6) -carboxyfluorescein diacetate, according to the manufacturer's protocol (Molecular Probes, Eugene, OR). Incubated with succinimidyl ester) and labeled [den Haan, Lehar et al., J. Exp. Med., 192, 1685-1695, 2000], K1735-WT cells were incubated for 3 days with or without addition and analyzed by FACS.

CTL Activity Assay : Two to four weeks after transplanting K1735-1D8, mice were sacrificed and spleen cell suspensions were prepared. If indicated, NK cells were removed using anti-Asialo GM1 antibody and rabbit complement (Cedaran, Ontario, Canada). 5 × 10 ⁶ spleen cells were incubated for 5 days with irradiated (12,000 rads) 1 × 10 ⁵ K1735-WT cells in 24-well plates (Costa Corporation, Cambridge, Mass.). Cytolytic activity was examined by a 4-hour Cr ⁵¹ release assay at various E / T ratios.

ELISPOT Assay : The murine IFN-γ ELISPOT kit (R & D Systems, Minneapolis, Minn.) Was used according to the manufacturer's protocol, and plates were counted by plate-scanning service (Cellular Technology, Ltd., Cleveland, Ohio).

Immunohistochemistry : Tissues were taken out 10-30 days after tumor injection, fixed in 10% formalin and sectioned at 4-6 μm, and the Vector ABC Kit (Vector Laboratories, Burlingham, CA, USA). )) Was stained using the manufacturer's protocol to detect CD4 + and CD8 + T cells. Sections were also stained with HE.

K1735-1D8 cells were rejected via mechanisms requiring CD4 + T cells and NK cells : Cell bound anti-4-1BB scFv was cloned into retroviral vector pLNCX (FIG. 15A). This construct was transfected to cells from the metastatic M2 clone of K1735, namely K1735-WT (Fidler and Hart, Cancer Res, 41, 3266-3267, 1981). The transfected cell line, K1735-1D8, expressed high levels of anti-4-1BB scFv on the surface (FIG. 15B).

K1735-WT cells continued to grow when subcutaneously transplanted into native syngeneic (C3H) mice. The same dose of K1735-1D8 cells initially formed a tumor with a surface area of approximately 30 mm ² , but it regressed and disappeared on day 20 (FIG. 15C). K1735-WT cells transfected with control vectors similarly constructed to encode anti-human CD28 scFv grew at the same rate as K1735-WT cells in C3H mice.

To investigate the role of NK cells as well as CD4 + and CD8 + T lymphocytes in the degeneration of K1735-1D8, MAb was injected intraperitoneally (ip) into natural mice to remove CD8 + T cells or CD4 + T cells, or both NK cells were removed using GM1 rabbit antibody in Asial. Control mice were injected with rat IgG. Twelve days later, splenocytes from similar mice were subcutaneously transplanted into each group when FACS analysis indicated that the targeted cell population was depleted. K1735-1D8 showed similar growth kinetics in mice injected with anti-CD8 MAb or control rat IgG, whereas K1735-1D8 was associated with K1735-WT when only CD4 + T cells were removed or both CD4 + T and CD8 + T cells were removed. It grew equally to the case. K1735-1D8 grew in all NK-depleted mice but grew more slowly than the CD4-depleted group (FIG. 15D).

Immunization with K1735-1D8 Induces Immunity with Memory and Specificity for K1735-WT : Ten mice / groups of C3H mice were implanted subcutaneously with K1735-1D8 or irradiated K1735-WT cells at 10-day intervals. The control group was injected subcutaneously with PBS. After 10 days, mice were injected with WT cells. K1735-1D8-immunized mice developed rejection against WT cells, but mice immunized with irradiated K1735-WT did not cause rejection (FIG. 16A). One immunization with K1735-1D8 cells was sufficient for protection against transplanted K1735-WT cells.

Two months after rejection against WT cells, rejection occurred when WT cells were transplanted back into mice immunized against K1735-1D8 (FIG. 16A). In contrast, cells from the antigenically irrelevant sarcoma Ag104 grew as was the case in the natural control “rejector” mice (FIG. 16B).

After two immunizations against K1735-1D8, skin dissipation occurred in approximately 20% of mice that rejected the transplanted WT cells, which persisted for over 4 months (FIG. 16C). There were no other signs of autoimmunity.

K1735-1D8 cells are effective as therapeutic vaccines : Three experiments were performed in which K1735-1D8 cells were transplanted into mice with established K1735-WT tumors. The first experiment was conducted with mice with subcutaneous tumors with a surface area of approximately 30 mm ² . In the first group, K1735-1D8 cells were injected four times at weekly intervals on the back side of the WT tumor. In the second group, irradiated K1735-WT cells were implanted, and in the third group, PBS was injected subcutaneously. WT tumors were grown in all control mice and in all mice immunized with irradiated K1735-WT cells. In contrast, they regressed in 4 out of 5 mice immunized with K1735-1D8 (FIG. 17A) and remained tumor free until the end of the experiment after 3 months and had no signs of toxicity. Tumor sinter nodes in the fifth mouse were reduced as much as in mice transplanted with K1735-1D8 cells.

In the second experiment, mice were injected subcutaneously with K1735-1D8 at weekly intervals, starting one day before or four or eight days after transplanting K1735-WT cells. At the same time, another group of mice received intraperitoneal (ip) injection of MAb 1D8 for comparison. The control group was injected intraperitoneally with PBS. As shown in Table 4, all control mice had to be sacrificed due to tumors of 100 mm ² or greater within 49 days of receiving WT cells. In contrast, all mice vaccinated with K1735-1D8 cells or injected with MAb 1D8 one day prior to transplantation of WT cells were tumor free at the end of the experiment 70 days after implantation of WT cells. Mice that began immunization against K1735-1D8 4 or 8 days after transplanting WT cells had no detectable tumors observed during the first 28 days, but after stopping vaccination, tumors in 4 out of 10 mice This occurred. Mice injected with MAb for the first time 8 days after implantation of WT cells developed tumors earlier than those of the corresponding K1735-1D8 group, but there was no difference in survival between the two groups. Tumors harvested 20 days after implantation of WT cells were sectioned, stained with H & E and evaluated by immunohistochemistry. Tumor sintered nodes from PBS control mice included a number of neoplastic cells and a few CD4 + and CD8 + T cells as in mice receiving MAb on day 8. In contrast, sintered sections from mice initially immunized against K1735-1D8 on day 8 contained a large number of CD4 + and CD8 + T lymphocytes and only a few neoplastic cells (FIG. 17B).

In the third experiment also lung metastasis was initiated by intravenous (iv) injection of 3 × 10 ⁵ K1735-WT cells into ⁵ mice / group. After 3 days, K1735-1D8 cells were implanted subcutaneously and this procedure was repeated for 1 month at weekly intervals; Control mice were injected with PBS. The experiment was terminated when one mouse died in the control group 37 days after receiving the WT cells. At this time, more than 500 metastatic lesions were present in the lungs of each control mouse, while fewer than 10 metastatic lesions were present in the lungs of immunized mice (FIG. 17C).

Immunization with K1735-1D8 Cells Induces Th1 Type Immune Responses : The proliferation rate of splenocytes from mice immunized against K1735-1D8, as measured by ³ [H] thymidine incorporation, was observed in natural mice or irradiated K1735-WT It was approximately twice the rate of spleen cell proliferation from mice immunized with (FIG. 18A). Splenocytes from K1735-1D8 immunized mice increased almost 4-fold when cultured with irradiated K1735-WT cells, but not when cultured with Ag104 cells. Spleen cells from natural mice and mice immunized against K1735-1D8 were also obtained from CFDA SE [den Haan, Lehar et al., J. Exp. Med., 192, 1685-1695, 2000], followed by incubation with or without irradiated K1735-WT cells for proliferation analysis. CD4 + and CD8 + splenic cells from K1735-1D8 immune mice proliferated actively (FIG. 18B) and maximized in the presence of K1735-WT cells. Splenocytes from natural mice did not proliferate.

ELISPOT analysis showed that a greater proportion of IFN-γ was produced in spleen cells from mice immunized against K1735-1D8 than splenocytes from native mice or mice bearing K1735-WT tumors (FIG. 19A). ELISPOT analysis of splenocytes from the experiments in Table 4 demonstrated reactivity in mice immunized with K1735-1D8 one day before or four days after transplantation of K1735-WT, which indicated that the splenocytes were first K1735- High when co-cultured with WT cells for 3 days (FIG. 19B). The highest reactivity in the group immunized 1 day prior to implantation of WT cells may be due to the smaller tumor size. Splenocytes or natural splenocytes from mice injected with anti-4-1BB MAb did not show reactivity.

Splenocytes from mice immunized against K1735-1D8 were incubated with irradiated K1735-WT cells for 5 days and then tested in a 4-hour Cr ⁵¹ release assay. If NK cells were not removed beforehand, K1735, Ag104 and YAC cells were well lysed to nearly equal levels (FIG. 19C). However, removal of NK cells by incubating spleen cells first with rabbit anti-Assial with GM1 antibody and complement showed significant levels of CTL activity against K1735-WT compared to Ag104 or YAC, but with anti- MHC class I MAb could be inhibited (FIG. 19D).

Demonstration of bystander effect when K1735-1D8 cells are mixed with K1735-WT cells : Tumor cells expressing anti-4-1BB scFv in vivo, for example by in vivo transfection, are also effective against wild type tumor cells. To investigate whether an immune response was induced, two experiments in which K1735-WT cells were mixed with K1735-1D8 cells were performed. The first experiment was performed by mixing two types of cells in equal numbers, in which case the tumors regressed after short-term in vivo growth. The second experiment was performed by mixing 2 × 10 ⁶ K1735-WT cells with 2 × 10 ⁵ K1735-1D8 cells. As shown in FIG. 20, the overgrowth of WT cells was inhibited when compared with the transplantation alone.

Thus, K1735-1D8 cells expressing cell-bound scFv from anti-4-1BB hybridoma 1D8 are rejected by syngeneic mice, and this rejection requires CD4 + T cells and NK cells rather than CD8 + T cells. It was. In addition, immunization against K1735-1D8 induced a systemic immune response with both memory and specificity for K1735-WT. In contrast, repeated immunization of mice with irradiated K1735-WT cells did not protect against WT cell injection. This is consistent with data that have been shown to have low immunogenicity even after K1735 expresses CD80. In vitro analysis showed that mouse spleen cells were immunized against K1735-1D8 cells. Vaccination of tumor-bearing mice had therapeutic efficacy both in tumors growing subcutaneously and in lungs.

Therapeutic efficacy observed for K1735-WT, a tumor with low immunogenicity and very low MHC class I expression, caused the tumor cells to be transfected to express anti- (human) 4-1BB scFv, which is indicated by cancer patients to conventional therapy. Support clinical trials used as autologous or allogeneic vaccines to destroy remaining metastases after receiving.

ScFv specific for human 4-1BB were generated from hybridoma 5B9 following the procedure described above for G19-4, 500A2 and 1D8 scFv. FIG. 21 shows the sequence of 5B9 scFv fused to human IgG1 hinge, CH2, and CH3 domains and to the transmembrane domain and cytoplasmic tail of human CD80.

See also the following references:

All patents, publications, scientific articles, websites, and other documents and references referred to or referred to herein are indicative of the level of skill in the art to which this invention pertains, and each of these references and references individually references its entirety. It is incorporated herein by reference to the same extent as referred to herein or as described herein in its entirety. Applicant reserves the right to actually introduce in this specification any and all documents and information from any such patents, publications, scientific articles, websites, electronically available information, and other references and documents.

Certain methods and compositions described herein are representative of preferred embodiments and are merely illustrative and are not intended to limit the scope of the invention. Other objects, aspects and embodiments will be apparent to those skilled in the art in view of this specification and are included within the spirit of the invention as defined in the following claims. It will be apparent to those skilled in the art that various changes and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention, which is suitably described herein by way of example, may be practiced without any element (s) or limitation (s) not specifically disclosed herein as essential. Thus, for example, any of the terms “comprising”, “consisting essentially of” and “consisting of” in each example herein, embodiment or example of the invention may be used interchangeably with the other two terms herein. . In addition, terms such as "comprising", "including", "containing", and the like will also be broadly interpreted and not limited. The methods and processes exemplarily suitably described herein may be practiced with a reversed order of steps and are not necessarily limited to the order as set forth herein or in the claims. Also, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural meanings unless the context clearly dictates otherwise. Thus, for example, reference to “host cells” includes a plurality of (eg, cultures or populations) such host cells. Under no circumstances should this patent specification be construed as limited to the specific examples, embodiments, or methods specifically disclosed herein. Under no circumstances is this patent specification to be construed as being limited to any reference made by any examiner or any other official or employee of the Office or Trademark Bureau, unless such reference is special and absolute or a reservation specifically adopted by the applicant in the opinion. It should not be.

The terms and expressions used are not limited to the terms used, and there is no intention to exclude any equivalents of the features or portions thereof indicated and described, but various modifications may be made within the scope of the present invention as claimed. It should be recognized that it can be applied. Thus, although the invention has been described in detail by the preferred embodiments and any features thereof, modifications and variations of the concepts disclosed herein will be readily apparent to those skilled in the art and such variations and modifications are also within the scope of the invention as defined in the appended claims. It should be understood that it is considered to belong.

The present invention has been described broadly and comprehensively. Each of the narrower and smaller classes belonging to the generic disclosure form part of the invention. Comprehensive descriptions of the invention are included unless there is a negative limitation that excludes any items from the description, whether or not the exclusions are specifically mentioned herein.

Other embodiments are included in the following claims. In addition, features or aspects of the invention will be described in terms of the Markush group, and those skilled in the art will recognize that the present invention is also described in terms of any individual member or member of a member belonging to the Markush group.

<110> LEDBETTER, JEFFREY       HAYDEN-LEDBETTER, MARTHA       HELLSTROM, INGEGERD       HELLSTROM, KARL ERIK <120> ACTIVATION OF TUMOR-REACTIVE LYMPHOCYTES VIA ANTIBODIES       OR GENES RECOGNIZING CD3 OR 4-1BB <130> 034474.0004 <140> 10 / 107,991 <141> 2002-04-26 <150> 60 / 286,585 <151> 2001-04-26 <160> 5 <170> PatentIn version 2.1 <210> 1 <211> 1687 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Mouse-Human Hybrid Gene <220> <221> CDS (222) (7) .. (1671) <220> <221> sig_peptide (222) (7) .. (75) <223> codes L6 Vkappa leader peptide flanked by Hind III and SalI sites <220> <221> V_region <222> (76) .. (406) <223> codes G19-4 mouse anti-human CD3 VL domain <220> <221> misc_feature 222 (406) .. (451) <223> codes (Gly4Ser) 3 linker <220> <221> V_region <451> (451) .. (816) <223> G19-4 mouse anti-human CD3 VH domain <220> <221> misc_feature (222) (816). (1521) <223> codes human IgG1 Fc domain (hinge, CH2, CH3) <220> <221> misc_feature (222) (1521) .. (1687) <223> codes human CD80 transmembrane and cytoplasmic domains and       attached restriction site <400> 1 aagctt atg gat ttt caa gtg cag att ttc agc ttc ctg cta atc agt 48        Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser          1 5 10 gct tca gtc ata atg tcc aga gga gtc gac atc cag atg aca cag act 96 Ala Ser Val Ile Met Ser Arg Gly Val Asp Ile Gln Met Thr Gln Thr  15 20 25 30 aca tcc tcc ctg tct gcc tct ctg gga gac aga gtc acc atc agt tgc 144 Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys                  35 40 45 agg gca agt cag gac att cgc aat tat tta aac tgg tat cag cag aaa 192 Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys              50 55 60 cca gat gga act gtt aaa ctc ctg atc tac tac aca tca aga tta cac 240 Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His          65 70 75 tca gga gtc cca tca agg ttc agt ggc agt ggg tct gga aca gat tat 288 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr      80 85 90 tct ctc acc att gcc aac ctg caa cca gaa gat att gcc act tac ttt 336 Ser Leu Thr Ile Ala Asn Leu Gln Pro Glu Asp Ile Ala Thr Tyr Phe  95 100 105 110 tgc caa cag ggt aat acg ctt ccg tgg acg ttc ggt gga ggc acc aaa 384 Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys                 115 120 125 ctg gta acc aaa cgg gag ctc ggt ggc ggt ggc tcg ggc ggt ggt ggg 432 Leu Val Thr Lys Arg Glu Leu Gly Gly Gly Gly Ser Gly Gly Gly             130 135 140 tcg ggt ggc ggc gga tct atc gat gag gtc cag ctg caa cag tct gga 480 Ser Gly Gly Gly Gly Ser Ile Asp Glu Val Gln Leu Gln Gln Ser Gly         145 150 155 cct gaa ctg gtg aag cct gga gct tca atg tcc tgc aag gcc tct ggt 528 Pro Glu Leu Val Lys Pro Gly Ala Ser Met Ser Cys Lys Ala Ser Gly     160 165 170 tac tca ttc act ggc tac atc gtg aac tgg ctg aag cag agc cat gga 576 Tyr Ser Phe Thr Gly Tyr Ile Val Asn Trp Leu Lys Gln Ser His Gly 175 180 185 190 aag aac ctt gag tgg att gga ctt att aat cca tac aaa ggt ctt act 624 Lys Asn Leu Glu Trp Ile Gly Leu Ile Asn Pro Tyr Lys Gly Leu Thr                 195 200 205 acc tac aac cag aaa ttc aag ggc aag gcc aca tta act gta gac aag 672 Thr Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys             210 215 220 tca tcc agc aca gcc tac atg gag ctc ctc agt ctg aca tct gaa gac 720 Ser Ser Ser Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp         225 230 235 tct gca gtc tat tac tgt gca aga tct ggg tac tat ggt gac tcg gac 768 Ser Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp     240 245 250 tgg tac ttc gat gtc tgg ggc gca ggg acc acg gtc acc gtc tcc tct 816 Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 255 260 265 270 gat ctg gag ccc aaa tct tct gac aaa act cac aca agc cca ccg agc 864 Asp Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser                 275 280 285 cca gca cct gaa ctc ctg ggg gga tcg tca gtc ttc ctc ttc ccc cca 912 Pro Ala Pro Glu Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro             290 295 300 aaa ccc aag gac acc ctc atg atc tcc cgg acc cct gag gtc aca tgc 960 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys         305 310 315 gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc aag ttc aac tgg 1008 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp     320 325 330 tac gtg gac ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag 1056 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 335 340 345 350 gag cag tac aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg 1104 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu                 355 360 365 cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag gtc tcc aac 1152 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn             370 375 380 aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggg 1200 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly         385 390 395 cag ccc cga gaa cca cag gtg tac acc ctg ccc cca tcc cgg gat gag 1248 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu     400 405 410 ctg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat 1296 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 415 420 425 430 ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac 1344 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn                 435 440 445 aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc 1392 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe             450 455 460 ctc tac agc aag ctc acc gtg gac aag agc agg tgg cag cag ggg aac 1440 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn         465 470 475 gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cac tac acg 1488 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr     480 485 490 cag aag agc ctc tcc ctg tct ccg ggt aaa gcg gat cct tcg aac ctg 1536 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ala Asp Pro Ser Asn Leu 495 500 505 510 ctc cca tcc tgg gcc att acc tta atc tca gta aat gga att ttt gtg 1584 Leu Pro Ser Trp Ala Ile Thr Leu Ile Ser Val Asn Gly Ile Phe Val                 515 520 525 ata tgc tgc ctg acc tac tgc ttt gcc cca aga tgc aga gag aga agg 1632 Ile Cys Cys Leu Thr Tyr Cys Phe Ala Pro Arg Cys Arg Glu Arg Arg             530 535 540 agg aat gag aga ttg aga agg gaa agt gta cgc cct gta taaatcgata 1681 Arg Asn Glu Arg Leu Arg Arg Glu Ser Val Arg Pro Val         545 550 555 ctcgag 1687 <210> 2 <211> 1696 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Mouse-Human Hybrid Gene <220> <221> CDS (222) (13680) (1680) <220> <221> sig_peptide (222) (13) .. (71) <223> codes 5B9 mouse anti-human 4-1BB VL leader peptide sequence <220> <221> V_region (222) (72) .. (412) <223> codes 5B9 mouse anti-human 4-1BB VL domain <220> <221> misc_feature <222> (413) .. (459) <223> codes (Gly4Ser) 3 linker peptide <220> <221> V_region <222> (460) .. (826) <223> codes 5B9 mouse anti-human 4-1BB VH domain <220> <221> misc_feature (827) .. (1527) <223> codes human IgG1 Fc domain (hinge, CH2, CH3) <220> <221> misc_feature (222) (1528) .. (1696) <223> codes human CD80 transmembrane and cytoplasmic domains with       attached restriction site <400> 2 aagcttgccg cc atg agg ttc tct gct cag ctt ctg ggg ctg ctt gtg ctc 51               Met Arg Phe Ser Ala Gln Leu Leu Gly Leu Leu Val Leu                 1 5 10 tgg atc cct gga tcc act gca gat att gtg atg acg cag gct gca ttc 99 Trp Ile Pro Gly Ser Thr Ala Asp Ile Val Met Thr Gln Ala Ala Phe      15 20 25 tcc aat cca gtc act ctt gga aca tca gct tcc atc tcc tgc agg tct 147 Ser Asn Pro Val Thr Leu Gly Thr Ser Ala Ser Ile Ser Cys Arg Ser  30 35 40 45 agt aag agt ctc cta cat agt aat ggc atc act tat ttg tat tgg tat 195 Ser Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr                  50 55 60 ctg cag aag cca ggc cag tct cct cag ctc ctg att tat cag atg tcc 243 Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser              65 70 75 aac ctt gcc tca gga gtc cca gac agg ttc agt agc agt ggg tca gga 291 Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Ser Ser Gly Ser Gly          80 85 90 act gat ttc aca ctg aga atc agc aga gtg gag gct gag gat gtg ggt 339 Thr Asp Phe Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly      95 100 105 gtt tat tac tgt gct caa aat cta gaa ctt ccg ctc acg ttc ggt gct 387 Val Tyr Tyr Cys Ala Gln Asn Leu Glu Leu Pro Leu Thr Phe Gly Ala 110 115 120 125 ggg acc aag ctg gag ctg aaa cgg ggt ggc ggt ggc tcg ggc ggt ggt 435 Gly Thr Lys Leu Glu Leu Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly                 130 135 140 ggg tcg ggt ggc ggc gga tcg tca cag gtg cag ctg aag cag tca gga 483 Gly Ser Gly Gly Gly Gly Ser Ser Gln Val Gln Leu Lys Gln Ser Gly             145 150 155 cct ggc cta gtg cag tcc tca cag agc ctg tcc atc acc tgc aca gtc 531 Pro Gly Leu Val Gln Ser Ser Gln Ser Leu Ser Ile Thr Cys Thr Val         160 165 170 tct ggt ttc tca tta act acc tat gct gta cac tgg gtt cgc cag tct 579 Ser Gly Phe Ser Leu Thr Thr Tyr Ala Val His Trp Val Arg Gln Ser     175 180 185 cca gga aag ggt ctg gag tgg ctg gga gtg ata tgg agt ggt gga atc 627 Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Ile 190 195 200 205 aca gac tat aat gca gct ttc ata tcc aga ctg agc atc acc aag gac 675 Thr Asp Tyr Asn Ala Ala Phe Ile Ser Arg Leu Ser Ile Thr Lys Asp                 210 215 220 gat tcc aag agc caa gtt ttc ttt aaa atg aac agt ctg caa cct aat 723 Asp Ser Lys Ser Gln Val Phe Phe Lys Met Asn Ser Leu Gln Pro Asn             225 230 235 gac aca gcc att tat tac tgt gcc aga aat ggg ggt gat aac tac cct 771 Asp Thr Ala Ile Tyr Tyr Cys Ala Arg Asn Gly Gly Asp Asn Tyr Pro         240 245 250 tat tac tat gct atg gac tac tgg ggt caa gga acc tca gtc acc gtc 819 Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val     255 260 265 tcc tct gat ctg gag ccc aaa tct tct gac aaa act cac aca agc cca 867 Ser Ser Asp Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro 270 275 280 285 ccg agc cca gca cct gaa ctc ctg ggg gga tcg tca gtc ttc ctc ttc 915 Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly Ser Ser Val Phe Leu Phe                 290 295 300 ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct gag gtc 963 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val             305 310 315 aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc aag ttc 1011 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe         320 325 330 aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca aag ccg 1059 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro     335 340 345 cgg gag gag cag tac aac agc acg tac cgt gtg gtc agc gtc ctc acc 1107 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 350 355 360 365 gtc ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag gtc 1155 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val                 370 375 380 tcc aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc aaa gcc 1203 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala             385 390 395 aaa ggg cag ccc cga gaa cca cag gtg tac acc ctg ccc cca tcc cgg 1251 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg         400 405 410 gat gag ctg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc 1299 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly     415 420 425 ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg 1347 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 430 435 440 445 gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc 1395 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser                 450 455 460 ttc ttc ctc tac agc aag ctc acc gtg gac aag agc agg tgg cag cag 1443 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln             465 470 475 ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cac 1491 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His         480 485 490 tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa gcg gat cct tcg 1539 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ala Asp Pro Ser     495 500 505 aac ctg ctc cca tcc tgg gcc att acc tta atc tca gta aat gga att 1587 Asn Leu Leu Pro Ser Trp Ala Ile Thr Leu Ile Ser Val Asn Gly Ile 510 515 520 525 ttt gtg ata tgc tgc ctg acc tac tgc ttt gcc cca aga tgc aga gag 1635 Phe Val Ile Cys Cys Leu Thr Tyr Cys Phe Ala Pro Arg Cys Arg Glu                 530 535 540 aga agg agg aat gag aga ttg aga agg gaa agt gta cgc cct gta 1680 Arg Arg Arg Asn Glu Arg Leu Arg Arg Glu Ser Val Arg Pro Val             545 550 555 taaatcgata ctcgag 1696 <210> 3 <211> 555 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Mouse-Human Fusion Protein <220> <221> SIGNAL (222) (1) .. (23) <223> L6 Vkappa signal peptide <220> <221> DOMAIN <222> (24) .. (133) <223> G19-4 mouse anti-human CD3 light chain variable domain <220> <221> PEPTIDE (222) (134) .. (148) (223) (Gly4Ser) 3 linker peptide <220> <221> DOMAIN (149) (149) .. (270) <223> G19-4 mouse anti-human VH domain <220> <221> MISC_FEATURE (222) (271) .. (504) <223> human IgG1 Fc domain (hinge, CH2, CH3) <220> <221> TRANSMEM (222) (505) .. (555) <223> human CD80 transmembrane domain and cytoplasmic tail <400> 3 Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser   1 5 10 15 Val Ile Met Ser Arg Gly Val Asp Ile Gln Met Thr Gln Thr Thr Ser              20 25 30 Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala          35 40 45 Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp      50 55 60 Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly 65 70 75 80 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu                  85 90 95 Thr Ile Ala Asn Leu Gln Pro Glu Asp Ile Ala Thr Tyr Phe Cys Gln             100 105 110 Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Val         115 120 125 Thr Lys Arg Glu Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly     130 135 140 Gly Gly Gly Ser Ile Asp Glu Val Gln Leu Gln Gln Ser Gly Pro Glu 145 150 155 160 Leu Val Lys Pro Gly Ala Ser Met Ser Cys Lys Ala Ser Gly Tyr Ser                 165 170 175      Phe Thr Gly Tyr Ile Val Asn Trp Leu Lys Gln Ser His Gly Lys Asn             180 185 190 Leu Glu Trp Ile Gly Leu Ile Asn Pro Tyr Lys Gly Leu Thr Thr Tyr         195 200 205 Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser     210 215 220 Ser Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala 225 230 235 240 Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr                 245 250 255 Phe Asp Val Trp Gly Ala Gly Thr Thr Thr Val Thr Val Ser Ser Asp Leu             260 265 270 Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala         275 280 285 Pro Glu Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro     290 295 300 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 305 310 315 320 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val                 325 330 335 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln             340 345 350 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln         355 360 365 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala     370 375 380 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 385 390 395 400 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr                 405 410 415 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser             420 425 430 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr         435 440 445 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr     450 455 460 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 465 470 475 480 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys                 485 490 495 Ser Leu Ser Leu Ser Pro Gly Lys Ala Asp Pro Ser Asn Leu Leu Pro             500 505 510 Ser Trp Ala Ile Thr Leu Ile Ser Val Asn Gly Ile Phe Val Ile Cys         515 520 525 Cys Leu Thr Tyr Cys Phe Ala Pro Arg Cys Arg Glu Arg Arg Arg Asn     530 535 540 Glu Arg Leu Arg Arg Glu Ser Val Arg Pro Val 545 550 555 <210> 4 <211> 556 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Mouse-Human Fusion Protein <220> <221> SIGNAL (222) (1) .. (20) <223> 5B9 mouse anti-human 4-1BB V kappa signal peptide <220> <221> DOMAIN (222) (21) .. (133) <223> 5B9 mouse anti-human 4-1BB VL domain <220> <221> PEPTIDE <222> (134) .. (149) (223) (Gly4Ser) 3 linker peptide <220> <221> DOMAIN (222) (150) .. (271) <223> 5B9 mouse anti-human 4-1BB VH domain <220> <221> MISC_FEATURE (272) .. (505) <223> human IgG1 Fc domain (hinge.CH2, CH3) <220> <221> TRANSMEM 222 (506) .. (555) <223> human CD80 trnasmembrane and cytoplasmic domains <400> 4 Met Arg Phe Ser Ala Gln Leu Leu Gly Leu Leu Val Leu Trp Ile Pro   1 5 10 15 Gly Ser Thr Ala Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro              20 25 30 Val Thr Leu Gly Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser          35 40 45 Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys      50 55 60 Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala  65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe                  85 90 95 Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr             100 105 110 Cys Ala Gln Asn Leu Glu Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys         115 120 125 Leu Glu Leu Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly     130 135 140 Gly Gly Gly Ser Ser Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu 145 150 155 160 Val Gln Ser Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe                 165 170 175 Ser Leu Thr Thr Tyr Ala Val His Trp Val Arg Gln Ser Pro Gly Lys             180 185 190 Gly Leu Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Ile Thr Asp Tyr         195 200 205 Asn Ala Ala Phe Ile Ser Arg Leu Ser Ile Thr Lys Asp Asp Ser Lys     210 215 220 Ser Gln Val Phe Phe Lys Met Asn Ser Leu Gln Pro Asn Asp Thr Ala 225 230 235 240 Ile Tyr Tyr Cys Ala Arg Asn Gly Gly Asp Asn Tyr Pro Tyr Tyr Tyr                 245 250 255 Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Asp             260 265 270 Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro         275 280 285 Ala Pro Glu Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys     290 295 300 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 305 310 315 320 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr                 325 330 335 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu             340 345 350 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His         355 360 365 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys     370 375 380 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 385 390 395 400 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu                 405 410 415 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro             420 425 430 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn         435 440 445 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu     450 455 460 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 465 470 475 480 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln                 485 490 495 Lys Ser Leu Ser Leu Ser Pro Gly Lys Ala Asp Pro Ser Asn Leu Leu             500 505 510 Pro Ser Trp Ala Ile Thr Leu Ile Ser Val Asn Gly Ile Phe Val Ile         515 520 525 Cys Cys Leu Thr Tyr Cys Phe Ala Pro Arg Cys Arg Glu Arg Arg Arg     530 535 540 Asn Glu Arg Leu Arg Arg Glu Ser Val Arg Pro Val 545 550 555 <210> 5 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 5 cgtcgatgag ctctagaatt cgcatgtgca agtccgatga gtcccccccc ccccc 55

Claims (24)

A culture system for producing tumor-reactive T lymphocytes comprising immobilized antibodies to T cells, antigen presenting cells, autologous or allogeneic tumor cells from a patient suffering from cancer and T cell receptors that induce polyclonal activation. The culture system of claim 1, wherein the antigen presenting cells are autologous monocytes differentiated in response to the T cells. The culture system of claim 1, wherein the autologous or allogeneic tumor cells express one or more genes that encode a molecule that is transfected to stimulate a direct or indirect T cell response. The culture system of claim 1, wherein the immobilized antibody binds to CD3 and CD28 receptors. A composition comprising a polynucleotide encoding an anti-CD3 scFv that can be expressed at the cell surface. The composition of claim 5, wherein the polynucleotide encodes G19-4 scFv. A composition comprising a polynucleotide encoding an anti-human 4-1BB scFv that can be expressed at the cell surface. 8. The composition of claim 7, wherein the polynucleotide encodes a 5B9 scFv. A composition comprising autologous or allogeneic tumor cells transfected with a gene encoding an anti-CD3 scFv that can be expressed at the cell surface. The composition of claim 9, wherein the autologous or allogeneic tumor cells are transfected with G19-4 scFv that can be expressed at the cell surface. A composition comprising autologous or allogeneic tumor cells transfected with a gene encoding an anti-4-1BB scFv that can be expressed at the cell surface. The composition of claim 11, wherein the autologous or allogeneic tumor cells are transfected with 5B9 scFv that can be expressed at the cell surface. A method of treating a subject suffering from cancer comprising administering to a subject suffering from cancer a tumor cell that has been transfected and expresses anti-CD3 scFv at the cell surface. The method of claim 13, wherein the tumor cells express G19-4. A method of treating a subject suffering from cancer comprising administering to a subject suffering from cancer a tumor cell that has been transfected and expresses anti-4-1BB scFv at the cell surface. The method of claim 15, wherein the tumor cells express 5B9. A method of treating a subject suffering from cancer comprising administering to a subject suffering from cancer a plasmid comprising a sequence encoding an anti-CD3 scFv that can be expressed at the cell surface. The method of claim 17, wherein the plasmid encodes G19-4 scFv. A method of treating a subject suffering from cancer comprising administering to a subject suffering from cancer a plasmid comprising a sequence encoding an anti-4-1BB that can be expressed on a cell surface. The method of claim 19, wherein the plasmid encodes 5B7 scFv. An isolated polynucleotide comprising a sequence encoding an anti-CD3 scFv that can be expressed at the cell surface. An isolated polynucleotide comprising a sequence encoding a G19-4 scFv that can be expressed at the cell surface. An isolated polynucleotide comprising a sequence encoding an anti-4-1BB that can be expressed at the cell surface. An isolated polynucleotide comprising a sequence encoding a 5B9 scFv that can be expressed at the cell surface.