Sherie L. Morrison
University of California, Los Angeles, CA

Use of Antibody Fusion Proteins as Therapeutic Agents

Malignant cells that have persisted to cause disease have failed to elicit an effective immune response. However, many studies have now demonstrated that it is possible to render tumor cells immunogenic by having them express immunostimulatory molecules such as cytokines and co-stimulatory molecules. Although the results obtained using genetically modified tumor cells have been promising, the ex vivo genetic modification and reintroduction of cells into patients is limited by its patient-specific nature and can be technically difficult, time consuming and expensive.

We have explored the alternative approach of using antibodies specific for tumor-associated antigens (TAAs) to direct the immunostimulatory molecules to the tumor microenvironment. The studies we have done have used antibodies specific for either the idiotype present on a murine lymphoma or HER2/neu expressed by murine carcinomas. Our approach is based on the concept that many different cell types and interactions are required for an effective immune response. To address these issues we have made antibody (Ab) fusions with the cytokines interleukin-2 (IL-2) (1-6), granulocyte/macrophage colony stimulating factor (GM-CSF) (7), and interleukin-12 (IL-12)(8,9), the chemokine RANTES (10), and the co-stimulatory molecule B7.1 (11).

Ab-IL-2 Fusion Proteins: Our attention has primarily been focused on the characterization of proteins in which IL-2 was fused after the end of CH3 (IgG3-CH3-IL2). This fusion protein resembles recombinant IL-2 (rIL-2) in its ability to stimulate proliferation of the IL-2 dependent cell line CTLL-2 but was more effective than rIL-2 in generating LAK cell activity (1). We found that IgG3-CH3-IL2 exhibits a significantly higher affinity than native IL-2 for the a chain of the high affinity IL-2 receptor (3). IgG3-CH3-IL2 has a half-life in mice of approximately 8 hours, which is 17 fold longer than the half-life reported for IL-2 by i.p. injection (2). IgG3-CH3-IL2 directed at the idiotype of the murine lymphoma 38C13 showed enhanced anti-tumor activity compared to the combination of Ab and IL-2 administered together (5). Similarly, anti-HER2/neu IgG3-(IL-2) was able to target human HER2/neu expressing cells and to support growth of the IL-2 dependent murine cell line CTLL-2. Treatment of immunocompetent mice with this Ab fusion protein resulted in significant retardation of the subcutaneous (s.c.) growth of CT26-HER2/neu tumors (6).

Ab-IL-12 Fusion Proteins: IL-12 is a disulfide-linked heterodimer of two subunits p35 and p40. Therefore, our strategy was to make a single chain version and for that purpose we used the cDNA coding for the murine fusion protein IL12.p40.L.Dp35 with the two subunits joined with a (Gly4Ser)3 linker (8). This protein showed a specific activity comparable to that of rIL-12 while a related construct with a different order of subunits (IL12.p35.L.Dp40) showed very low activity. IL-12 was fused to the amino terminus of the anti-HER2/neu-IgG3. At this position, proteins that require N-terminal processing or folding for activity such as nerve growth factor (NGF) remain active (12). We showed that the presence of IL-12 at this position did not interfere with the ability of the antibody to bind HER2/neu. Anti-HER2/neu IL12-IgG3 binds the IL-12 receptor and resembles recombinant IL-12 in its ability to stimulate proliferation of peripheral blood lymphocytes. It also showed anti-tumor efficacy and was most effective when administered to established tumors. The mechanism of the anti-tumor activity exhibited by anti-HER2/neu IL12-IgG3 is highly complex and involves a combination of T- and NK-cell activity, a switch to a TH1 immune response and anti-antiogenic activity (9).

Ab-(GM-CSF) Fusion Proteins: Anti-HER2/neu containing granulocyte/macrophage colony-stimulating factor (GM-CSF) was able to target HER2/neu expressing cells and to support growth of a GM-CSF dependent murine myeloid cell line, FDC-P1 and to activate the J774.2 macrophage. It was comparable to the parental Ab in its ability to effect ADCC mediated tumor cell lysis (7). Pharmacokinetic studies showed that the half-life of anti-HER2/neu IgG3-(GM-CSF) depended on the injected dose with longer in vivo persistance observed at higher doses. Biodistribution studies showed that anti-HER2/neu IgG3-(GM-CSF) is mainly localized in the spleen. In addition, anti-HER2/neu IgG3-(GM-CSF) was able to target the HER2/neu expressing murine tumor CT26-HER2/neu and enhance both TH1 and TH2 mediated immune responses. Treatment with this Ab fusion protein resulted in significant retardation in the growth of s.c. CT26-HER2/neu tumors.

Fusion Proteins as Adjuvants for Vaccines: We have recently investigated the use of antibody fusion proteins as adjuvants for vaccination with tumor associated antigens. Mice were vaccinated with the extracellular domain of HER2/neu (ECD-HER2) either by itself or in combination with anti-HER2/neu IgG3-(GM-CSF), anti-HER2/neu IL12-IgG3 or anti-HER2/neu IgG3-(IL-2) and challenged with a syngeneic carcinoma expressing the rat HER2/neu (TUBO). There was a significant retardation of tumor growth and long-term survivors in mice vaccinated with ECD-HER2 plus all three antibody fusion proteins as compared to the control groups. An anti-(ECD-HER2) humoral immune response was detected in all vaccinated groups with ECD-HER plus Ab-(GM-CSF) and ECD-HER2 plus Ab-(IL-2) vaccinated mice showing the highest titers with significant production of both IgG1 and IgG2a antibodies. Ab-(IL-12) vaccinated mice showed increased IgG2a Abs but not IgG1 Abs. Immune sera showed significant in vitro anti-proliferative activity against the human breast cancer line SK-BR-3 with the level of inhibition correlated with the anti(ECD-HER2) level. Overall our results suggest that both humoral and cell-mediated response may contribute to the observed anti-tumor activity.

Future Directions: Our studies have shown that fusion proteins in which an Ab binding specificity that recognizes a TAA is joined to an immunostimulatory molecule potentiate the ability of animals to inhibit the growth of tumors and as effective adjuvants for vaccination. However, using a single protein it was not possible to achieve complete rejection of the growing tumors. Therefore the goal of ongoing research is to use combinations of fusion proteins with different immune effector molecules to determine if it is possible to achieve a more potent immune response that will result in complete rejection of growing tumors.

References:

1. Harvill, E.T. and Morrison, S.L., Immunotechnology, 1:95-105, 1995.
2. Harvill, E.T, Fleming, J.M. and Morrison, S.L., J. Immunol., 157:3165-3170, 1996.
3. Harvill, E.T. and Morrison, S.L., Molecular Immunology, 33:1007-1014, 1996.
4. Penichet, M.L., Harvill, E. and Morrison, S.L., Human Antibodies, 8:106-118, 1997.
5. Penichet, M.L., Harvill, E. and Morrison, S.L., J. of Interferon and Cytokine Research,
18:597-607, 1998.
6. Penichet, M. L., et. al., Human Antibodies, 10:43-49, 2001.
7. Dela Cruz, J.S., et. al., J. Immunol. 165:5112-5121, 2000.
8. Peng, L.S., Penichet, M.L., and Morrison, S.L., J. Immunol., 163:250-258, 1999.
9. Peng, L., et. al., J. of Interferon and Cytokine Research, 21:709-720, 2001.
10. Challita-Eid, P.-M., et. al., J. Immunol., 161:3729-3736, 1998.
11. Challita, P.-M., et. al., J. Immunol., 160:3419-3426, 1998.
12. McGrath, J.P., et. al., J. Neuroscience Research 47:123-133, 1997.