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Sep 7, 2011

Researchers Demonstrate Viability of Targeting Intracellular Proteins with Antibodies to Treat Cancer

  • Antibodies targeting intracellular oncogenic proteins can effectively be used to halt cancer growth in tumor-bearing mice, scientists claim. They also showed that vaccinating cancer-bearing animals with antigens derived from the same intracellular oncoproteins is similarly effective in triggering an immune response against the cancer cells.

    Qi Zeng, Ph.D., and colleagues, at the Agency for Science, Technology and Research (A*Star), say antigen-induced vaccination may be particularly useful in genetically based familial cancers, for which treatment could be used to prime the immune system of a family member against the target oncoporotein. They report their work in a paper titled “Targeting Intracellular Oncoproteins with Antibody Therapy or Vaccination” and published in Translational Medicine.

    The development of antibody therapies is focused largely on targeting extracellular or cell surface proteins rather than intracellular targets because antibodies are too large to be taken up into cells, Drs. Zeng et al. explain. However, since the late 1970s a number of studies have suggested that antibodies can be taken up into cells and that this may be a common phenomenon in autoimmune diseases.

    The authors claim their results document “a proof of concept showing that antibody or antigen-induced antibody therapies against intracellular proteins can specifically and significantly arrest tumor progression in vivo.”

    While the mechanisms underpinning the therapeutic activity of both antibody and antigen vaccination approaches remains unclear, a number of possibilities can be envisaged, they continue. One is that a small fraction of intracellular antigens is released due to necrosis or cancer cell lysis. Alternatively, some of the intracellular antigens may be externalized and displayed on the surface of cancer cells by unconventional secretion, enabling the antibodies to bind and trigger immune responses such as ADCC.

    A third possibility is that the antibodies are taken up by the cancer cells in an antigen-specific manner and that their interaction with the intracellular targets leads to cancer cell apoptosis; this would be akin to the cellular phenomenon previously described for autoantibodies, the researchers point out. This third possibility is supported by the finding that B cells can enhance cancer cell uptake of antibodies in coculture systems.

    The likelihood is that a combination of several mechanisms, possibly including complement-mediated events, are actually involved in achieving the final therapeutic consequence of antibodies against intracellular oncoproteins, they conclude. “With cancer treatment becoming more individualized, the ability to target a whole new list of intracellular oncoproteins previously thought to be untargetable by therapeutic antibodies or vaccinations can expand the scope for tailor-made cancer therapy as well as usher in a new era of tailor-made cancer vaccines.”

    To see whether antibody therapy could be used to target intracellular cancer proteins, the researchers set up separate experiments targeting PRL-3 (phosphatase of regenerating liver 3), enhanced green fluorescent protein (EGFP), and the polyolmavirus middle T (mT) oncoprotein.  

    The team had previously demonstrated the effectiveness of antibody therapy against intracellular PRL proteins in a metastatic tumor model using immunocompromised nude mice that lack T cells. EGFP was chosen as the second target because although it is not expressed naturally in host tissues, EGFP serves as an artificial cancer-specific intracellular protein when expressed in cancer cells. The mT protein, meanwhile, is carried by MMTV-PymT transgenic mice that spontaneously develop breast tumors and are widely used as a spontaneous tumor model.

    The researchers also hypothesized that if an antibody can recognize an intracellular antigen, then an intracellular antigen could potentially be used as a vaccine to naturally trigger antibody production by the host immune system, achieving a similar effect as antibody therapy. Indeed, they suggest, “such an approach would be fairly inexpensive and yet effective in treating pre-existing cancer or preventing cancer from developing.”

    To investigate this possibility they also explored the use of the three purified proteins, PRL-3, EGFP, and mT, as therapeutic vaccines to block cancer formation in animal models that express the oncoproteins. The team’s first experiment tested a PRL-3-targeting immunoglobulin in immunocompetent wild-type C57BL/6 mice that had previously been injected with melanoma cells expressing high levels of endogenous PRL-3 (F0).

    While untreated melanoma-bearing control mice demonstrated severe weight loss and metastatic tumors in multiple organs, the F0 animals treated using the PRL-3 antibody appeared healthier and consistently gained body weight. Specificity of the antibody to PRL-3 was confirmed by treating animals carrying melanomas that expressed very low PRL-3 (F10). These animals all succumbed to cancer whether they were treated with the PRL-3 antibody or not.

    Because the team’s previous studies had shown that the PRL-3 mAb could inhibit  tumor formation in nude mice lacking T cells, the team investigated whether B lymphocytes are involved in the beneficial effects of antibody treatment. They used the PRL-3 antibody to treat PRL-3-expressing (F0) melanoma in an engineered strain of immunodeficient Rag-2 mice that lack mature B and T lymphocytes.

    Interestingly, both untreated and PRL-3 antibody-treated animals demonstrated equivalent tumor burdens, indicating the antibody was not effective.  And when they used the antibody to treat a strain of PRL-3 melanoma-bearing mice that generate T cells but lack mature B cells (muMT animals), there was again no significant benefit of treatment on tumor burden or lifespan.  These combined results suggest that antibody therapy required mature B cell function in the host for antitumor activity.

    Previous work by Dr. Zeng’s team had shown that nonpermeabilized cancer cells expressing PRL-3 (or PRL-1) can specifically take up their respective antibodies in vitro. Because their latest studies showed that B lymphocytes were required to boost the efficacy of PRL-3 antibody therapy, the next step was to evaluate the effect of B cells on the internalization of the PRL-3 antibody in both the PRL-3 expressing melanoma cell line (F0) and the in F10 melanoma cells that don’t express PRL-3.

    They found that coculturing F0 cancer cells with isolated B cells increased by 5- to 6-fold the percentage of cells taking up the antibody from 13%  to 75%. It didn’t, however, have any effects on the PRL-3 nonexpressing cells. “This result suggests that a possible role of host B cells is to facilitate and enhance antibody uptake by the cancer cells,” the team remarks.

    Having used the PRL-3 antibody to demonstrate the feasibility of using an antibody against an endogenous intracellular cancer target, the researchers moved on to test the approach against other intracellular proteins, using EGFP as an example. EGFP was exogenously overexpressed as an artificial cancer-specific intracellular protein in F0 and F10 melanoma cancer cell lines, and pooled EGFP-F0 and EGFP-F10 cells were sorted to enrich for EGFP-positive cells. The resulting pooled cells still contained about 20% non-EGFP-expressing cells, which served as an internal negative control.

    Mice were injected with either the EGFP-F0 or EGFP-F10 cells to establish tumors and subsequently treated with an EGFP mAb. As predicted, both EGFP-F0 and EGFP-F10 cells responded equally well to EGFP antibody treatment in terms of tumor regression (i.e. regardless of PRL-3 expression), but the beneficial effects were only seen in the EGFP-expressing tumors.

    Tumors formed from the F0 or F10 cells that didn’t express EGFP weren’t affected by antibody therapy. “This further supports that targeting intracellular protein with antibody therapy depends on specific antibody-antigen interactions for therapeutic efficacy,” the researchers add.

    With the demonstration that antibody therapy could work against both endogenous (PRL-3) and exogenous (EGFP) intracellular proteins, the team tested the effects of antibody therapy in the MMTV-PymT TG mouse model of spontaneous mammary tumorigenesis. Animals were injected with an mT antibody at six weeks of age, and twice again over the next six weeks.

    Compared with the untreated mice, treated animals demonstrated far smaller tumor sizes and more normal growth and development of breast tissue. Lifespan was also increased in the treated cohort. “In all, we found that 100% (26 of 26) of untreated mice carried marked breast tumors and multiple lung tumors, whereas only 16.6% (3 of 18) of mT antibody-treated mice developed tumor-bearing breasts, and most of the treated mice showed marked reduction in the formation of metastatic breast tumors when examined at about 12 to 13 weeks,” Dr. Zeng and colleagues report.

    The next question to answer was whether intracellular proteins could be used as antigens to challenge and trigger an immune response that generates natural antibodies against the target intracellular proteins. Eight-week-old C57BL/6 mice were treated using multiple doses of either the PRL-3 antigen or EGFP protein, and the immunized mice then divided into two groups and intravenously injected with either EGFP-F0 or EGFP-F10 melanoma cells.

    Compared with nonimmunized mice , those vaccinated using PRL-3 displayed reduced metastatic tumors formed by EGFP-F0 cancer cells in numerous tissues but weren’t able to block the tumors formed by EGFP-F10 melanoma cells that express low PRL-3 protein. “Again, this suggests that antigen-induced, host-produced PRL-3 antibodies have an impact on inhibiting PRL-3-expressing EGFP-F0 (but not EGFP-F10) tumors,” the researchers write. In contrast, EGFP immunization reduced metastatic tumors formed by both EGFP-F0 and EGFP-F10 cancer cells in multiple tissues because both cell lines express EGFP.

    The team finally tested the vaccination strategy in the MMTV-PymT TG spontaneous mammary tumor model. The results were striking. Compared with nonimmunized animals, those vaccinated with the mT antigen developed high anti-mT antibody titres, exhibited a marked reduction in the weight of mammary gland tumors, and survived much longer. In fact, all the nonimmunized mice carried marked breast tumors, but only 23.5% of mT-immunized mice developed tumor-bearing breasts.


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