Tumor infiltrating lymphocytes (TILs) in a colorectal carcinoma. Source: Nephron. http://bit.ly/QdusBi

On April 27, 2011 we published an article on this blog entitled “Adoptive immunotherapy for metastatic melanoma?” This blog post, which was in part based on an article in the April 2011 issue of The Scientist, described a treatment for metastatic melanoma known as adoptive cell transfer (ACT), or adoptive immunotherapy. ACT is the only type of therapy that has resulted in high percentages of durable compete responses in metastatic melanoma. A durable complete response, which is tantamount to a cure, is the real desire of every cancer patient, and of their loved ones, and of caring physicians who treat them.

In ACT, a physician/researcher extracts a patient’s antigen-specific immune cells, which are usually found in tumor tissue. Such cells are known as “tumor infiltrating lymphocytes” (TILs). He or she then expands the numbers of the antitumor T lymphocytes in cell culture, using the T-cell growth factor, IL-2. The physician/researcher then infuses the cells, plus IL-2, intravenously into the patient. The infused T cells traffic to tumors and can mediate their destruction. Prior to TIL infusion, the patient may have his or her immune system temporarily ablated via “preparative lymphodepletion” with chemotherapy and sometimes also total-body irradiation. The preparative lymphodepletion treatment is associated with enhanced persistence of the transferred TILs.

In a clinical study of ACT published in 2011, the treatment resulted in the disappearance of all tumors in 20/93 patients (21.5%) with advanced metastatic melanoma. For 19 of these 20 patients (95%), the complete responses have been durable and long-lasting, in some cases lasting for over 7 years. (See also the Faculty of 1000 evaluation.)

Research on the mechanistic basis of adoptive immunotherapy, as well as on means to improve ACT technologies, is ongoing, so there is the potential to improve the durable complete response rate further. We featured a December 2012 Nature cancer immunotherapy review article that included a discussion of ways to improve ACT in the 2011 end-of-year article on our Biopharmconsortium Blog.

Despite the fact that ACT is the only type of therapy that has resulted in high percentages of durable compete responses in metastatic melanoma, it is not widely available. ACT is only available in a small number of cancer canters worldwide, and there has been little commercial interest in developing ACT.

Adoptive immunotherapies are still considered experimental, are not FDA-approved, and are not covered by third party payers. Thus only a handful of locations can bear the financial burden of administering adoptive immunotherapy. If a cancer center has a cell production facility with the required staff, the cost of producing a single dose of T-cells for adoptive transfer is approximately $20,000. ACT treatment also entails factoring in the cost of hospitalization. However, most patients only require a single dose.

The cost of ACT is, however, much lower than a full course of other immunotherapies, such as the dendritic cell vaccine Provenge (which is not indicated for melanoma) or the immunotheraputic MAb drug ipilimumab, both of which cost approximately $120,000. The total cost of a 6-month treatment with the targeted kinase drug vemurafenib is $56,400. None of these treatments result in durable complete responses, except in a very small number of patients.

The main problem with increasing the availability of ACT is the lack of a viable business model for its commercialization. Adoptive immunotherapies lack a clearly defined claim to intellectual property (IP), since the patient’s own cells are not a “drug” to be patented. It would be difficult for a private company to pursue clinical trials for FDA approval and commercialization of ACT. To conduct such trials, a company would need to build a specialized cell processing and treatment facility, with a highly trained and competent staff. If the therapies cannot be protected as IP, and would therefore not be considered proprietary, it would not be worth the effort and expense to commercialize them.

The Novartis/Penn agreement

Now comes an agreement (announced on August 6, 2012) between Novartis and the University of Pennsylvania (Penn) aimed at commercializing adoptive cellular immunotherapy.

The agreement is based on one of the improvements to ACT discussed in the December 2011 Nature cancer immunotherapy review, in which autologous T cells isolated from patient blood (not from tumors) are engineered with retroviral vectors carrying chimeric antigen receptors (CARs). This technology allows physician researchers to extend ACT beyond patients from whom TILs can be isolated and expanded. It also enables them to extend ACT beyond melanoma to include other types of solid tumors and leukemias and lymphomas. Unlike TILs, CAR-bearing T cells do not recognize surface antigens on tumor cells [presented by major histocompatibility complex (MHC) proteins] via their T-cell receptors. They instead recognize surface proteins on tumor cells via the affinity domain on the engineered CAR. This also expands the kinds of tumor cells that can be recognized, as compared to TILs.

In the Penn studies, led by David L. Porter, M.D. at the Perelman School of Medicine of the University of Pennsylvania, the researchers used this technology to treat patients with chronic lymphocytic leukemia (CLL). They designed a lentiviral vector expressing a chimeric antigen receptor with specificity for the B-cell antigen CD19, coupled with the T cell costimulatory receptor CD137 and CD3-zeta (a signal-transduction component of the T-cell antigen receptor) signaling domains. They used this vector to engineer autologous T cells, and infused the engineered cells into the patient after preparative lymphodepletion with chemotherapy. In a pilot study with one patient with refractory chronic lymphocytic leukemia (CLL), the infused cells exhibited in vivo expansion and anti-leukemia activity. The treatment resulted in complete remission, which was ongoing 10 months after initiation.

In a later study, the researchers treated three more patients with autologous engineered CAR T cells. The T cells expanded over 1000-fold in vivo, trafficked to bone marrow, and continued to express CARs at high levels for at least six months. The CAR T-cells showed anti-leukemia activity, with each engineered T cell eliminating approximately 1000 CLL cells. A CD19-specific immune response was demonstrated in the blood and bone marrow of two of three patients; these patents showed complete remission. Some of the cells in these patients persisted as memory CAR T cells and retained anti-CD19 effector activity. These results suggested that this technology has the potential to effectively treat B cell malignancies, and to induce durable complete remissions in at least a portion of patients.

As reported in August 2012, of the three patients who showed positive results with the anti-CD19 immunotherapy, two were still in complete remission over a year into the CART-19 trial, and the third patient maintained partial remission for more than seven months. An immune deficiency resulting from the treatment known as hypogammaglobulinemia, an expected chronic toxic effect of anti-B cell therapy, was corrected with infusions of intravenous immune globulin. Patients were also treated for symptoms associated with tumor lysis syndrome, an effect of tumor breakdown.

Under the agreement, Novartis acquired exclusive rights from Penn to CART-19, the investigational CAR immunotherapy that was the focus of the studies discussed earlier. The target of CART-19, CD19, is associated with several B-cell malignancies, including CLL, B-cell acute lymphocytic leukemia and diffuse large B-cell lymphoma. Novartis expects to initiate a Phase II clinical trial with CART-19 in collaboration with Penn during the fourth quarter of 2012.

To facilitate the discovery and development of additional types of CAR immunotherapy, Novartis and Penn will build the Center for Advanced Cellular Therapies (CACT) at Penn. This center will be established specifically to develop and manufacture adoptive T-cell immunotherapies under the research collaboration between Penn and Novartis.

Penn also granted Novartis an exclusive worldwide license to CARs developed through the collaboration for all indications, in addition to CART-19. In return, Novartis will provide an up-front payment, research funding, funding for the establishment of the CACT and milestone payments for the achievement of certain clinical, regulatory and commercial milestones as well as and royalties on any sales.

Business implications of the Novartis/Penn agreement

The feasibility of developing and commercializing CAR T-cell-based immunotherapy is based on the ability of Penn to patent and license its CAR technology. Such an approach in principle would apply to immunotherapies based on other types of engineered T cells, such as those engineered with retroviral vectors carrying cloned T-cell receptors, as discussed in the December 2011 Nature review article.

As discussed earlier, adoptive immunotherapies with engineered T cells would also address patients with a variety of types of cancer (not just melanoma) and from who TILs cannot be isolated. However, whether any therapies with engineered T cells can give the percentages of durable complete responses seen with TIL-based therapy of melanoma remains to be demonstrated in clinical trials.

The Novartis/Penn agreement represents an example of Novartis’ willingness to take risks, in order to “bring innovative therapies to patients”, as stated by Hervé Hoppenot, President, Novartis Oncology. Mark Fishman, President of the Novartis Institutes for BioMedical Research, sees cancer immunotherapy as “one of the exciting frontiers in cancer research,” and the CAR technology as showing “early promise as a new way for treating cancer.”

Novartis thus has not built a viable business model for TIL-based ACT. However, it is developing a parallel technology that is more protectable than TILs, which might result in bringing adoptive cellular immunotherapy to a much larger number of patients.

BiTE immunotherapy

Meanwhile another type of T-cell-based immunotherapy technology (also discussed in the Nature review) is now under development. This is bi-specific T-cell engager (BiTE) technology, originally developed by the German-American biotech company Micromet. Amgen acquired Micromet in April 2012, and is now developing the first BiTE agent, blinatumomab. Blinatumomab is a bispecific MAb that binds to CD19 on target B-cell malignancies and to CD3 (an invariant component of the T-cell receptor) on T cells. This results in the activation of the T cell to exert cytotoxic activity on the target cell. BiTE immunotherapy does not require isolation and culture of autologous T cells, and BITE technology and therapeutics derived from it are patentable as with other drugs.

In May 2012, Amgen reported that blinatumomab treatment gave a high rate (72 percent) of complete responses in a Phase 2 study in patients with relapsed or refractory B-precursor acute lymphoblastic leukemia (ALL). The rate of remission seen in this trial was a great improvement over the current standard of care. However, no durable complete responses were seen; median survival was 9 months.

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As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or an initial one-to-one consultation on an issue that is key to your company’s success, please contact us by phone or e-mail. We also welcome your comments on this or any other article on this blog.

Macrophages attack a cancer cell

An article in the June 2012 issue of OncologyLive, authored by the publication’s senior editor, Anita T. Shaffer, reviews cancer immunotherapies now in late-stage clinical trials, and discusses the prospects for the field.

The article begins with a discussion of the recent renaissance of cancer immunotherapy, as exemplified by the April 2010 FDA approval of Dendreon’s Sipuleucel-T (APC8015, Provenge) and the March 2011 FDA approval of Ipilimumab [Medarex/Bristol-Myers Squibb’s (BMS’) Yervoy]. It then went on to discuss the exciting Phase 1 results with Medarex/BMS’ anti-PD-1 MAb, which we featured in the June 28, 2012 article on the Biopharmconsortium Blog.

But the bulk of the article was a discussion of the current late-stage (Phase 3) active immunotherapy pipeline. The article’s table lists 14 such agents. If one eliminates Cel-Sci/Teva’s Multikine (which is a mixture of cytokines), that leaves 13 agents, at least most of which can be described as therapeutic cancer vaccines. These products range from dendritic cell vaccines to tumor cell-based vaccines and viruses that encode tumor antigens.

For example, Argos Therapeutics‘ AGS-003 (Arcelis) is an autologous dendritic cell vaccine loaded with the patient’s own messenger RNA (mRNA). This vaccine is in Phase 3 clinical trials in patients with newly diagnosed metastatic renal cell carcinoma (mRCC). We mentioned Argos and its technology in our November 25, 2011 article on the late Ralph Steinman, MD, who had discovered the dendritic cell and elucidated its central role in the immune system. Dr. Steinman was a cofounder of Argos. Patient mRNA in Argos’ cellular immunotherapy product encode tumor antigens, which are expressed on the surface of the dendritic cells. The dendritic cells then potentiate the production of tumor antigen-specific T cells which attack the patient’s tumor.

According to a July 2 2012 company news release, AGS-003 is a fully personalized immunotherapy that preferentially targets mutated tumor antigens, which drive disease progression. Patient T cells recognize these antigens as foreign. This enables AGS-003 to direct a specific and potent anti-tumor immune response, without attacking normal tissues.

In a Phase 2 study of a combination of AGS-003 and sunitinib (Pfizer’s Sutent, the standard of care for mRCC), researchers demonstrated a statistically significant correlation between the number of anti-tumor T cells induced and overall survival in mRCC patients receiving AGS-003. Adding AGS-003 to sunitinib doubled overall survival for these patients compared to historical results for unfavorable risk patients treated with sunitinib alone. Over 50 percent of patients in the study survived longer than 30 months after initiating therapy, which is four times the expected rate for sunitinib.

Another type of cancer vaccine is based on modified cancer cells. In our Steinman article, this strategy is represented by BioSante’s GVAX cancer vaccines [now licensed by Aduro BioTech (Berkeley, CA)]. One such vaccine, GVAX Pancreas for pancreatic cancer (which is now in clinical trials) is based on human pancreatic cancer cell lines that have been engineered to secrete the immunostimulant granulocyte-macrophage colony-stimulating factor (GM-CSF), and have then been lethally irradiated. Since the most advanced GVAX products are in Phase 1 and Phase 2 clinical trials, GVAX was not covered in the OncologyLive article.

However, other more advanced immunotherapies, such as NewLink Genetics‘ HyperAcute Pancreas cancer immunotherapy (in Phase 3 trials), also consist of modified cancer cells. HyperAcute Pancreas consists of equal parts of two separate allogeneic pancreatic cancer cell lines engineered to express α-galactosidase (an enzyme that is not expressed by natural human pancreatic tumors).

Another type of cancer vaccine is based on viruses that encode tumor antigens. For example, Bavarian Nordic A/S’ PROSTVAC, a treatment for prostate cancer, is a  sequentially dosed combination of vaccinia and fowlpox poxviruses that encode an altered, more immunogenic form of prostate-specific antigen (PSA) plus three immune enhancing costimulatory molecules ( B7.1, ICAM-1, and Lfa-3).

The late-stage immunotherapies listed in the table in the OncologyLive article include cancer vaccines that represent several design strategies other than the three mentioned here.

Some good news about sipuleucel-T

The OncologyLive article also referred to an abstract presented at the 2012 American Society of Clinical Oncology (ASCO) meeting, which suggests that the survival advantage for prostate cancer patients treated with sipuleucel-T was significantly greater than the 4.1-month benefit reported in the Phase 3 trial that led to approval of the agent. The analysis reported in this abstract indicates that the overall survival treatment benefit with sipilleucel-T ranged from 4.1 months to  7.8 months.

Conclusions

As illustrated by the number of late-stage cancer immunotherapies in development, as well as the approval of two drugs in 2010 and 2011, cancer immunotherapy is here to stay. One question in the use of such immunotherapies, as highlighted in the OncologyLive article, is how they will be integrated with such established modalities as cytotoxic chemotherapy, radiation therapy, and targeted cancer therapies.

Another factor is cost. A course of treatment with sipuleucel-T costs $93,000, and the cost of a course of treatment with ipilimumab is $120,000. However, as pointed out in the OncologyLive article, the total cost of treatment with other modalities that may continue for months or years may be higher. Nevertheless, the cost of cancer therapies, especially those that only increase overall survival by a few months, is a great concern to patients, physicians, and payers.

It must be remembered, however, that nearly all cancer therapies, when first introduced to the market, gave only slightly enhanced survival over older treatments. However, as oncologists learned how to use the therapies better (e.g., with changes in dosing, use in other groups of cancer patients, and/or use in combination therapies), numerous therapies eventually gave long-term remissions or even cures and proved to be cost-effective indeed.

Another issue with the cancer immunotherapy field, as pointed out in the OncologyLive article, is the difficulty of raising capital for cancer immunotherapy specialty companies. This is especially true in the current market, where most biotech companies have difficulty in raising capital. However, what venture capitalists and Big Pharma consider to be “premature technologies” or “unproven” emerging early-stage areas, as is usually the case, have particular difficulty in attracting investment.

Nevertheless, if and when additional late-stage cancer immunotherapy agents successfully complete Phase 3 trials and gain approval, this may demonstrate to investors that cancer immunotherapy has graduated from the premature-technology stage. In that case, cancer immunotherapy specialty companies may find it easier to attract capital, and large pharmaceutical companies may wish to acquire some of these companies. Since Big Pharma already is involved in developing such immunotherapies as anti-PD-1 and anti PD-1L, and ipilimumab is already a marketed Big Pharma drug, that should not be much of a stretch.

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As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to  your company, please click here. We also welcome your comments on this or any other article on this blog.

The American Society of Clinical Oncology (ASCO) held its 2012 Annual Meeting on June 1-5, 2012. Arguably the highlight of the meeting was the June 2, 2012 presentation by Bristol-Myers Squibb (BMS) on its Phase 1 immunotherapeutic, anti-PD-1 (BMS-936558). The results of this study were also published ahead of print on June 2, in the online version of the New England Journal of Medicine. Nature published a “News in Focus” article on the same subject by Nature staff writer Erika Check Hayden in its 6 June issue.

BMS acquired its anti-PD-1 MAb product BMS-936558 (MDX-1106) via its 2009 acquisition of Medarex. This is the same way in which BMS acquired its now-marketed immunotherapy, ipilimumab (Yervoy), which was approved by the FDA in March 2011. Both BMS-936558 and ipilimumab are monoclonal antibodies (MAbs). Ono Pharmaceuticals has been a partner in the development of anti-PD-1 MAb since its original collaboration with Medarex; Ono retains the right to exclusively develop and market the agent (which is also designated as ONO-4538) in Japan, Korea and Taiwan.

PD-1 (“programmed cell death-1”) is a receptor on the surface of activated T lymphocytes of the immune system. PD-1 is a member of the CD28/CTLA4 family of T cell regulators. Like CTLA4, the target of ipilimumab, PD-1 is a negative regulator of T-cell receptor signals. When a protein on the surface of some tumor cells, known as PD-1 ligand (PD-L1), binds to PD-1 on T cells that recognize antigens on these tumors cells, this results in the blockage of the ability of the T cells to carry out an anti-tumor immune response. Anti-PD-1 MAb binds to PD-1 on T cells, thus preventing PD-L1 on tumor cells from binding to the PD-1 and initiating an inhibitory signal. Anti-tumor T cells are then free to initiate immune responses against the tumor cells. This mechanism of action is completely analogous to that of ipilimumab, which binds to CTLA4 and thus prevents negative signaling from that molecule.

Phase 1 clinical study of Medarex/BMS’s anti-PD-1

The Phase 1 clinical study was carried out by a multi-institution team of investigators, led by Suzanne L. Topalian, M.D. (Johns Hopkins University School of Medicine, Baltimore, MD.) The researchers enrolled patients with advanced melanoma, non-small-cell lung cancer (NSCLC), prostate cancer, renal cell cancer (RCC), or colorectal cancer. Patients received anti-PD-1 at a dose between 0.1 and 10.0 milligrams per kilogram of body weight every two weeks. Tumor response was determined after each 8-week treatment cycle. Patients received up to 12 cycles of treatment until either unacceptable adverse events, disease progression or a complete response occurred. A total of 296 patients received treatment through February 24, 2012.

Among the 236 patients in whom tumor responses could be evaluated, objective responses were observed in patients with NSCLC, melanoma, or RCC. Cumulative response rates (among patients treated with all doses of anti-PD-1) were 18% among patients with NSCLC, 28% among patients with melanoma, and 27% among patients with RCC.  These responses were durable–20 of 31 responses lasted 1 year or more in patients with 1 year or more of follow-up. Anti–PD-1 produced objective responses in approximately one in four to one in five patients with NSCLC, melanoma, or RCC.

In addition to patients with objective responses, other patients treated with anti-PD-1 exhibited stable disease lasting 24 weeks or more–5 patients (7%) with NSCLC, 6 patients (6%) with melanoma, and 9 patients (27%) with RCC.

Significant drug-related adverse effects were seen in 11% of the patients, including three deaths due to pulmonary toxicity. In most cases, adverse effects were reversible, and the observed adverse-event profile does not appear to preclude the use of the drug. A maximum tolerated dose was not reached in this study.

The exciting finding of this study is that anti-PD-1 produced durable responses not only in melanoma and RCC (the two types of cancer that are deemed to be “immunogenic”), but also in NSCLC, a much more common cancer that kills more people per year than any other cancer. Moreover, response rates with anti-PD-1 were much higher that those achieved with the other recently approved immunotherapeutics. In the Phase 3 clinical trial of ipilimumab that led to its approval, this drug gave response rates of 11% in melanoma patients. The other recently approved immunotherapeutic, the prostate cancer-specific dendritic cell vaccine Sipuleucel-T (Dendreon’s Provenge, APC8015), gives very low response rates and no complete responses. According to Antoni Ribas (Jonsson Comp­rehensive Cancer Center, University of California, Los Angeles CA) as quoted Ms. Hayden’s Nature “News in Focus” review, if an immunotherapy “breaks the 10% ceiling” as did anti-PD-1, it becomes “even more important and clinically relevant”.

Despite the exciting efficacy results with anti-PD-1, and despite the fact that it was deemed that the adverse-event profile did not appear to preclude the use of the drug, researchers would still like to get away from the serious adverse effects (including three deaths) seen with anti-PD-1. As with other immunotherapeutics (e.g., ipilimumab), researchers hypothesize that anti-PD-1’s serious adverse effects were due to autoimmune responses.

Phase 1 clinical study of Medarex/BMS’ anti-PD-L1

A potential way of achieving similar efficacy to anti-PD-1 with an improved safety profile is provided by another Phase 1 immunotherapeutic,  anti-PD-L1. Anti-PD-L1 MAb drugs are being developed by Medarex/BMS, Roche/Genentech, and other companies. As mentioned earlier, PD-L1 is the binding partner of PD-1 that is expressed on some tumor cells. As quoted in the Nature “News in Focus” review, Ira Mellman (vice-president of research oncology at Genentech), believes that anti-PD-L1 might have fewer adverse effects than anti-PD-1. That is because anti-PD-L1 would target tumor cells while leaving T cells free to participate in immune networks that work to prevent autoimmune reactions.

The results of a Phase 1 clinical study of BMS/Medarex’ anti-PD-L1 (also known as MDX-1105) were also published ahead of print in the online version of the New England Journal of Medicine on June 2, 2012; this was a “companion study” to the Phase 1 study of anti-PD-1. This study was also carried out by a multi-institution team of investigators, led by Julie R. Brahmer, M.D. (Johns Hopkins University School of Medicine, Baltimore, MD.); Dr. Topalian, among other investigators on the anti-PD-1 trial, also participated in the study.

This Phase 1 trial was a dose escalation study that was carried out via a similar protocol to the anti-PD-1 trial discussed earlier. As of February 24, 2012, a total of 207 patients — 75 with NSCLC, 55 with melanoma, 18 with colorectal cancer, 17 with RCC, 17 with ovarian cancer, 14 with pancreatic cancer, 7 with gastric cancer, and 4 with breast cancer — had received anti–PD-L1 antibody, for a median duration of 12 weeks. Among patients with an evaluable response, an objective response (i.e., a complete or partial response) was seen in 17% of patients with melanoma, 12% of patients with RCC, 10% of patients with NSCLC, and 6% of patients with ovarian cancer. Responses lasted for 1 year or more in 8 of 16 patients with at least 1 year of follow-up. Prolonged disease stabilization was seen in 12-41% of patients with advanced cancers, including NSCLC, melanoma, and RCC.

Significant drug-related adverse effects were seen in 9% of patients.

Although the two agents were not compared directly in a randomized trial, the frequency of objective responses for anti–PD-L1 MAb appears to be somewhat lower than that observed for anti–PD-1 MAb in initial Phase 1 trials; the frequency and severity of significant drug-related adverse events also appears to be lower. However, whether these differences will hold up in Phase 2 and 3 clinical trials remains to be determined. The clinically appropriate dose of anti–PD-L1 will also require further definition later studies. Nevertheless, the Phase 1 trial showed that anti-PD-L1 MAb induced durable tumor regression (objective response rate of 6-17%) and prolonged disease stabilization (rate of 12-41% at 24 weeks) in patients with select advanced cancers, including NSCLC, a tumor type that had been deemed to be “non-immunogenic”. This is essentially the same result that was observed for anti-PD-1MAb.

A predictive biomarker for treatment with anti-PD-1?

As with other modes of cancer therapy, it would be very useful to have mechanism-based predictive biomarkers to identify appropriate candidates for treatment with anti-PD-1 or anti-PD-L1 immunotherapy. The findings of the Phase 1 anti-PD-1 study suggest that PD-L1 expression in tumors is a candidate biomarker that warrants further evaluation for use in selecting patients for immunotherapy with anti–PD-1 MAb. The researchers found that 36% of patients with PD-L1–positive tumors achieved an objective response, while no patients with PD-L1–negative tumors achieved such a response. These results suggest that PD-L1 expression on the surface of tumor cells in pre-treatment tumor specimens may be associated with an objective response. However, further studies will be necessary to define the role of PD-L1 as a predictive biomarker of response to anti–PD-1 therapy. Similarly, it appears reasonable that tumor expression of PD-L1 may be a predictive biomarker of response to anti-PD-L1 therapy. However, this hypothesis must also be tested in further clinical studies.

Further studies of anti-PD-1 MAb

Two studies of BMS-936558/MDX-1106 anti–PD-1 MAb, both in advanced/metastatic clear-cell RCC, are now recruiting patients. One trial is a Phase 1 biomarker study involving immunologic and tumor marker correlates of efficacy (progression-free survival and tumor response). The other trial is a Phase 2 efficacy (progression-free survival and tumor response) study; this is a dose ranging study that is designed to determine if a dose response exists. Phase 3 studies of BMS-936558/MDX-1106 anti–PD-1 MAb for the treatment of non–small-cell lung cancer, melanoma, and renal-cell cancer are also being planned.

Conclusions

The exciting results of the studies with BMS’ anti-PD-1 and anti-PD-L1 have only been in Phase 1 studies. Thus caution is advisable in interpreting these results, pending the results of further clinical studies. Nevertheless, these results, together with the recent approval of ipilimumab (Medarex/Bristol-Myers Squibb’s Yervoy) and of Sipuleucel-T (Dendreon’s Provenge), indicate that cancer immunotherapy, a field that not so long ago was regarded as an impractical dream, is very much alive and well. In addition to clinical development and approval of immunotherapeutic agents, exciting basic and drug discovery research in this field is ongoing. This was recognized by the awarding of the 2011 Nobel Prize in Physiology or Medicine for research with profound implications for the development of cancer immunotherapies.

The Biopharmconsortium Blog has been covering new developments in cancer immunotherapy since the spring of 2011. Our earlier articles on this subject (with links) are listed in our December 31, 2011 article, entitled “Read the cancer immunotherapy review in the 22 December 2011 issue of Nature!”

Cancer immunotherapy represents one of several “scientifically premature” or “frontier science” areas discussed in this blog that are providing new opportunities for drug discovery and development–for young entrepreneurial biotech start-ups and for more established biotechnology and pharmaceutical companies. Corporate strategists would do well to explore such areas for potential new R&D programs for their companies.

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As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to  your company, please click here. We also welcome your comments on this or any other article on this blog.

RAS/BRAF/PI3K pathways. Source: Source BioScience

Two previous articles on this blog have included discussions of the “co-clinical mouse/human trial” strategy for improving mouse models of human cancer, and simultaneously improving human clinical trials of drugs for these cancers. Now comes an article on the use of a co-clinical trial strategy in personalized treatment of non-small cell lung cancer (NSCLC) in the 29 March 2012 issue of Nature. In the same issue of Nature is a News and Views article by Genentech’s Leisa Johnson Ph.D. that provides a minireview of the research article.

As we discussed in our April 15, 2010 article on this blog, the co-clinical trial strategy has been developed by Pier Paolo Pandolfi, MD, PhD (Director, Cancer and Genetics Program, Beth Israel-Deaconess Medical Center Cancer Center and the Dana-Farber/Harvard Cancer Center) and his colleagues.

As discussed in that article, these researchers constructed genetically engineered transgenic mouse strains that have genetic changes that mimic those found in human cancers. These mouse models spontaneous develop cancers that resemble the corresponding human cancers. In Dr. Pandolfi’s  ongoing co-clinical mouse/human trial project, researchers simultaneously treat a genetically engineered mouse model and patients with tumors that exhibit the same set of genetic changes with the same experimental targeted drugs. The goal of this two-year project is to determine to what extent the mouse models are predictive of patient response to therapeutic agents, and of tumor progression and survival. The studies may thus result in validated mouse models that are more predictive of drug efficacy than the currently standard xenograft models.

The human clinical trials being “shadowed” by simultaneous studies in mice included Phase 3 trials of several targeted therapies for lung and prostate cancer. Xenograft models in which tumor tissue from the patients had been transplanted into immunosuppressed mice were also being tested in parallel with the genetically engineered mouse models. This project represents the most rigorous test to date of how well genetically engineered mouse models of cancer can predict clinical outcomes.

Our October 28, 2011 blog article, which is mainly a review of a 29 September 2011 Nature article by Nature writer Heidi Ledford, Ph.D., focuses on ways to fix the clinical trial system. Our article includes a discussion of a co-clinical trial published in January 2011. This trial utilized two genetically-engineered PDGF (platelet-derived growth factor)-driven mouse models of the brain tumor glioblastoma multiforme (GBM), one of which had an intact PTEN gene and the other of which was PTEN deficient. In this trial, researchers tested the Akt inhibitor perifosine (Keryx Biopharmaceuticals, an alkylphospholipid) and the mTOR inhibitor CCI-779 (temsirolimus; Pfizer’s Torisel), both alone and in combination, in vitro and in vivo. The drugs and drug combinations were tested in cultured primary glioma cell cultures derived from the PTEN-null and PTEN-intact mouse PDGF-driven GBM models, and in the animal models themselves.

The studies showed that both in vitro and in vivo, the most effective inhibition of Akt and mTOR activity in both PTEN-intact and PTEN-null cells in animals was achieved by using both inhibitors in combination.  In vivo, the decreased Akt and mTOR signaling seen in mice treated with the combination therapy correlated with decreased tumor cell proliferation and increased cell death; these changes were independent of PTEN status. The co-clinical animal study also suggested new ways of screening GBM patients for inclusion in clinical trials of treatment with perifosine and/or CCI-779.

The new co-clinical trial reported in the March 2012 issue of Nature

The March 2012 Nature report describes research carried out by a large, multi-institution academic consortium, which included Dr. Pandolfi. It focuses on strategies for treatment of patients with non-small-cell lung cancer (NSCLC) with activating mutations in KRAS (Kirsten rat sarcoma viral oncogene homolog). These mutations occur in 20–30% of NSCLC cases, and patients whose tumors carry KRAS driver mutations have a poor prognosis. Moreover, KRAS is a “hard” or “undruggable” target, and no researchers have thus been able to discover inhibitors of oncogenic KRAS.

Because of the intractability of oncogenic KRAS as a target, researchers have been attempting to develop combination therapies for mutant-KRAS tumors (including, for example, colorectal cancers as well as NSCLCs) that address downstream pathways controlled by KRAS. We discussed examples of these strategies in our book-length report Multitargeted Therapies: Promiscuous Drugs and Combination Therapies, published by Cambridge Healthtech Institute/Insight Pharma Reports in 2011. Strategies discussed in that report are based on the finding that KRAS controls signal transduction via two key pathways: the B-Raf-MEK-ERK pathway and the PI3K-Akt pathway. This is illustrated in the figure at the top of this article. As discussed in our 2011 report, researchers are attempting to develop treatments of mutant-KRAS tumors that involve combination therapies with an inhibitor of the mitogen-activated protein kinase (MEK) together with an inhibitor of phosphatidylinositol 3-kinase (PI3K). Researchers are also attempting to develop combination therapies of MEK inhibitors with standard cytotoxic chemotherapies, which if successful will avoid having to use combinations of two expensive targeted therapies.

In the co-clinical trial that is the focus of the 29 March 2012 Nature research report and News and Views commentary, researchers developed a genetically-engineered mouse model to study treatment of mutant-KRAS NSCLCs with either the antimitotic chemotherapy drug docetaxel alone, or docetaxel in combination with the MEK kinase inhibitor selumetinib (AZD6244, AstraZeneca). In the parallel human clinical trial, researchers are also studying treatment of patients with mutant-KRAS NSCLC with docetaxel alone or docetaxel plus selumetinib. (There is no treatment arm in the human clinical trial in which patients are treated with selumetinib alone, since selumetinib monotherapy of NSCLC patients had shown no efficacy in a previous Phase 2 study; this was confirmed in mouse model studies.)

In humans with mutant-KRAS NSCLC, many tumors with mutations in KRAS have concomitant genetic alterations in other genes that may affect response to therapy. Therefore, the co-clinical trial researchers wished to design mouse models with lung tumors with either Kras mutations alone or with mutations in both Kras and another gene that is often concomitantly mutated in mutant-KRAS NSCLCs in humans. The researchers therefore constructed mouse models with cancers bearing the activating Kras(G12D) mutation, either alone or together with an inactivating mutation in either p53 or Lkb1. The researchers achieved this via a conditional mutation system using nasal instillation of specifically genetically-engineered adenoviruses. As result, a small percentage of lung epithelial cells harbored these mutations. It is from these cells that the NSCLC-like tumors arose, analogous to the clonal origin of sporadic lung tumors in humans.

Of the two tumor suppressor genes that are frequently mutated in human mutant-KRAS NSCLCs and that were modeled by the co-clinical trial researchers, p53, often called the “guardian of the genome”, is familiar to most of you. The other gene, Lkb1 [liver kinase B1, also known as serine/threonine kinase 11 (STK11)], was discussed in an earlier article on the Biopharmconsortium Blog, entitled “The great metformin mystery–genomics, diabetes, and cancer.”

LKB1 (whether in regulation of gluconeogenesis in the liver or in its role as a tumor suppressor) acts by activation of AMPK (AMP-activated kinase, a sensor of intracellular energy status.) In lung cancer (as shown by the same group that performed the 2012 co-clinical trial), LKB1 acts to modulate lung cancer differentiation and metastasis.  Germline mutations in LKB1 are associated with the familial disease Peutz-Jegher syndrome, in which patients develop benign polyps in the gastrointestinal tract. Studying a mouse model of mutant-LKB1 Peutz-Jeger syndrome, Reuben J. Shaw (Howard Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, CA, who was prominently mentioned in our “great metformin mystery” article) and his colleagues showed that the LKB1-AMPK pathway downregulates the mTOR pathway–specifically the rapamycin-sensitive mTOR complex 1 (mTORC1) and its downstream effector hypoxia-inducible factor-1α (HIF-1α). HIF-1α expression in turn upregulates the expression of its downstream effectors hexokinase II and glucose transporter 1 (GLUT1), which are involved in cellular utilization of glucose. LKB1-deficient polyps in this mouse model thus show increased expression of hexokinase II and GLUT1, resulting in dramatically increased glucose utilization.

In the new co-clinical trial, genetically-engineered mice that showed established lung tumors [as determined via magnetic resonance imaging (MRI)] were randomized to receive either docetaxel, selumetinib, or a combination of the two drugs. For tumors with only a Kras mutation, treatment with docetaxel alone resulted in a modest rate of response, with 30% of mice showing a partial response. Mice that bore mutant-Kras tumors that also had mutations in either p53 or Lkb1 had much lower rates of response to docetaxel monotherapy (5% and 0%, respectively), and more of these mice showed progressive disease on MRI or died of their disease. Of mice treated with the docetaxel/selumetinib combination, those with single-mutant Kras tumors showed a 92% overall response rate, and those with mutant Kras/p53 tumors showed a 61% overall response rate. However, mice with mutant Kras/Lkb1 cancers showed only a modest response to the docetaxel/selumetinib combination; 33% of mice achieved a partial response. The difference in response rate of mice with Kras/Lkb1 tumors to docetaxel/selumetinib compared to the other two genotypes was found to be statistically significant.

Using the genetically-engineered NSCLC mouse model in biomarker development

In human patients in clinical trials or in treatment for their cancers, performing repeated biopsies to monitor treatment is difficult. The co-clinical trial researchers therefore wished to develop less invasive means of monitoring both co-clinical and clinical trials of docetaxel/selumetinib treatment of NSCLC. They therefore tested the use of positron emission tomography (PET) with 18F-fluoro-2-deoxyglucose (FDG-PET) as an indicator of early response to therapy that could be used in the clinic.  Prior to its radioactive decay (109.8 minute half -life), 18F-FDG is a nonmetabolizable glucose analogue that moves into cells that is preferentially taken up by high-glucose utilizing cells. The researchers found that both Kras/p53 and Kras/Lkb1 tumors showed higher FDG uptake in vivo in the mouse model than did single-mutant Kras tumors. As expected from the earlier study, GLUT1 expression was elevated in Kras/Lkb1 mutant tumors. In human patients, pre-treatment, mutant KRAS/LKB1 tumors also showed a higher FDG uptake that did KRAS tumors negative for LKB1.

Treatment of the mice with docetaxel alone gave no significant changes in FDG uptake in Kras, Kras/p53, or Kras/Lkb1 tumors in vivo. However, within 24 hours of the first dosing of docetaxel/selumetinib, FDG uptake was markedly inhibited in Kras and Kras/p53 tumors. In contrast, treatment of mice with Kras/Lkb1 mutant tumors gave no appreciable decrease in FDG uptake in these tumors. These results show that early changes in tumor metabolism, as assessed by FDG-PET, predict antitumor efficacy of docetaxel/selumetinib treatment.

The FDG-PET study in this mouse model supports the use of this imaging method as a biomarker to monitor the course of treatment in humans.

Cellular signaling in mutant Kras, Kras/p53, and Kras/Lkb1 tumors

The researchers assessed activation of relevant intracellular pathways in tumors in treated and untreated mice with mutant Kras, Kras/p53, and Kras/Lkb1 lung cancers. They performed these studies using two different methods–immunostaining of cancer nodules for phosphorylated ERK, and immunoblotting of tumor lysates. In untreated mice, Kras/Lkb1 tumors show less activation of ERK than do Kras and Kras/p53 tumors, with Kras/p53 tumors showing the greatest amount of activation of the MEK-ERK pathway. Docetaxel had no discernible effect on signaling via the MEK-ERK pathway. Selumetinib alone resulted in decreased ERK activation in Kras and Kras/p53 tumors, but there was still residual activity. The docetaxel/selumetinib combination, however, was more effective in eliminating ERK activation. Pharmacokinetic studies indicated that selumetinib levels were higher in both serum and tumors of mice treated with docetaxel/selumetinib that in those treated with selumetinib alone; this might account for the more potent suppression of MEK-ERK signaling by the combination therapy as compared to selumetinib monotherapy. The researchers studied MEK-ERK activation (as determined by phospho-ERK staining) in  a set of 57 human NSCLC tumors with known RAS, p53 and LKB1 mutation status. As with the tumors in the mouse model, of seven patients whose tumors harbored the KRAS activating mutation, the three patients with concurrent p53 mutations showed higher levels of ERK activation.

The decreased activation of ERK in Kras/Lkb1 tumors suggested that these tumors utilize other pathways to drive their proliferation. On the basis of their prior studies of signal transduction in mutant-Lkb1 lung tumors, the researchers focused on AKT and SRC. Immunoblotting studies showed that Kras/Lkb1 mutant tumors had elevated activation of both AKT and SRC. As one can see from the figure at the top of this article, AKT is a downstream effector of PI3K; since the PI3K/AKT pathway regulates expression of GLUT1 and hexokinase, increased activation of the PI3K/AKT pathway is consistent with the increased uptake of FDG of mutant Kras/Lkb1 tumors. In the figure, SRC (which is not shown) represents one of the major “other effectors” controlled by RAS. These results indicate that concomitant mutation of Lkb1 in mutant-Kras NSCLCs may shift the signaling pathways that drive tumor proliferation from MEK-ERK to PI3K/AKT and/or SRC. This shift would result in primarily resistance of Kras/Lkb1 tumors to treatment with docetaxel/selumetinib.

Long-term benefits of treatment of mice with mutant-Kras and Kras/p53 tumors with docetaxel/selumetinib as opposed to docetaxel monotherapy

The researchers studied long-term treatment of mice with mutant-Kras and Kras/p53 tumors with docetaxel monotherapy versus docetaxel/selumetinib. In mice with mutant-Kras tumors, treatment with docetaxel monotherapy gave stable disease for several weeks, while docetaxel/selumetinib treatment resulted in tumor regression and slower regrowth of tumors. The addition of selumetinib to docetaxel significantly prolonged progression-free survival in these mice. In mice with Kras/p53 tumors, treatment with docetaxel alone resulted in progressive disease, but docetaxel/selumetinib treatment resulted in initial disease regression followed by progression, resulting in prolonged progression-free survival. These results indicate that treatment with combination therapy as opposed to docetaxel alone results in improved progression-free survival, but not cure, in mice with Kras– and Kras/p53-mutant tumors.

The researchers also investigated mechanisms of acquired tumor resistance in mice with mutant-Kras and Kras/p53 tumors, which had been treated long-term with docetaxel/selumetinib. In moribund animals that had received this treatment, all tumor nodules examined showed a recurrence of ERK phosphorylation. This suggested that acquired resistance could be at least in part due to reactivation of MEK–ERK signaling despite ongoing treatment with selumetinib. Evaluation of resistant tumor nodules suggested that more than one mechanism for pathway reactivation was occurring; study of these mechanisms is ongoing.

Conclusions of the new co-clinical study

The results of this co-clinical study predict that docetaxel/selumetinib combination therapy will be more effective than docetaxel monotherapy in several sub-classes of mutant-KRAS NSCLC. This prediction is consistent with the early results of a Phase 2 clinical trial of these two drug combinations in second-line treatment of patients with KRAS-mutant NSCLC.

However, the co-clinical trial also predicts that concurrent mutation of LKB1 in mutant-KRAS  tumors will result in primary resistance to docetaxel/selumetinib combination therapy, perhaps via activation of parallel signaling pathways such as AKT and SRC. Since LKB1 status is not being prospectively assessed in the ongoing human clinical trial, the presence of patients with cancers having concurrent LKB1 mutations may diminish the differences between treatment arms based solely on KRAS status. The results of the co-clinical trial suggests that researchers perform retrospective analysis of p53 and LKB1 status in samples from the concurrent human clinical trial. Future clinical trials should then be designed that involve prospective analysis to ensure sufficient enrollment of patients with all three genotypes to enable sufficiently powered sub-group analyses.

Although the results of the co-clinical trial indicate that patients with mutant KRAS/LBK1 tumors be excluded from trials of docetaxel/selumetinib treatment, the research group that has been conducting the co-clinical trial has also been conducting studies that may lead to treatment strategies for KRAS/LBK1 tumors.

The co-clinical trial also allowed researchers to design and validate biomarker strategies, specifically the potential use of the less-invasive FDG-PET to predict efficacy and to monitor treatment. The co-clinical animal-model study also enabled the discovery of mechanisms of both primary and acquired resistance that might benefit future clinical trials and discovery/development of drugs. (The studies on acquired resistance are in a early stage and are ongoing). Any mechanisms of acquired resistance discovered in co-clinical studies should be confirmed in human clinical trials by examining biopsy samples from patients who relapse on therapy. The ability to assess mechanisms of resistance in preclinical or co-clinical animal studies may enable researchers to design rational drug combination strategies that can be implemented in future clinical studies.

The results of the new co-clinical trial strengthens the contention that co-clinical trials in genetically-engineered mice can provide data that can predict the outcome of parallel human clinical trials. Co-clinical trials can also be used to generate new hypotheses for use in analyzing concurrent human trials, and for design of future clinical studies. Moreover, co-clinical trials can result in the validation of improved animal models for human cancers, which can be used in research and preclinical testing of oncology agents, and in validation of biomarkers for clinical studies in oncology. Given the inadequacy of “standard” xenograft models, which is a major factor in the high attrition rate of pipeline oncology drugs, the availability of validated genetically-engineered animal models may be expected to enable improved oncology drug development.

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As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to  your company, please click here. We also welcome your comments on this or any other article on this blog.

Eltrombopag

On April 13, 2012, Informa’s Scrip Insights the publication of a new book-length report, Advances in the Discovery of Protein-Protein Interaction Modulators, by Allan B. Haberman, Ph.D.

Protein-protein interactions (PPIs) are of central importance in biochemical pathways, including pathways involved in disease processes. However, PPIs have been considered the prototypical “undruggable” or “challenging” targets. The discovery of small-molecule drugs that can serve as antagonists or agonists of PPIs, and which are capable of being successfully taken into human clinical trials, has been extremely difficult. Among the theoretical reasons for this is that contact surfaces involved in PPIs are usually large and flat, and lack the types of cavities present in the surfaces of proteins that bind to small-molecule ligands.

Nevertheless, over the last twenty years, researchers have developed a set of technologies and strategies that have enabled them, in a several cases, to discover developable small-molecule PPI modulators. One direct PPI agonist, the thrombopoietin mimetic eltrombopag (Ligand/GlaxoSmithKline’s Promacta/Revolade), has reached the market. The chemical structure of this compound is illustrated in the figure above. Several other small-molecule PPI modulators are in clinical trials. Despite this progress, the discovery and development of small-molecule PPI modulators has been one-at-a-time, slow and laborious.

The new strategic importance of protein-protein interactions as drug targets

Meanwhile, PPIs as potential drug targets have acquired a key strategic importance for the success of the pharmaceutical industry. Over at least the last decade, pharmaceutical R&D has failed to develop enough high-valued new drugs to make up for or exceed revenues from blockbusters that are losing patent protection. As we have discussed in previous publications and in , this low productivity is mainly due to pipeline attrition. There are several factors (ranging from target selection through drug design, preclinical studies, identification and use of biomarkers, and design of clinical trials) that can influence pipeline attrition.

However, increasing numbers of industry leaders and analysts identify target selection as the key factor that is limiting the productivity of pharmaceutical R&D. For example, I served as a workshop leader at Hanson Wade’s   last summer, which took that point of view. There are at least several such conferences throughout the year, which are organized at the request of industry leaders.

Industry experts who identify poor target selection as a major cause of pharma R&D’s productivity woes conclude that the main issue is that companies are running out of “druggable” targets that have not already been addressed by marketed drugs. As of 2011, only 2% of human proteins have been targeted with drugs. Most of the remaining disease-relevant proteins, including transcription factors and many other types of signaling proteins, work via interacting with other proteins in PPIs. Therefore, in order to reverse its R&D slump, the pharmaceutical industry needs to develop technologies and strategies to address PPIs and other hitherto “undruggable” targets.

Contents of the report

Our report discusses technologies and strategies that enable the discovery of drugs targeting PPIs, including both small-molecule and synthetic peptidic modulators. It includes case studies on the discovery of compounds that address specific target classes, with emphasis on agents that have reached human clinical studies. This includes addressing the issue of the need to produce PPI modulatory agents that have pharmacological properties that will enable them to be good clinical candidates.

The report also includes discussions of second-generation technologies for the discovery of small-molecule and peptidic PPI modulators, which have been developed by such companies as Forma, Ensemble, and Aileron, and by academic laboratories. The field of PPI modulator discovery has represented a “premature technology”, i.e., a field of biomedical science in which consistent practicable therapeutic applications are in the indefinite future, due to difficult technological hurdles. We have discussed premature technologies on earlier on this blog. The second-generation technologies are designed to overcome the hurdles and to thus enable a more accelerated and systematic approach to PPI drug discovery and development.

In part as the result of the development of these technologies, and of the increasing strategic importance of PPI modulator development, companies have been moving into the field. Examples include Bristol-Myers Squibb, Pfizer, Novartis, and Roche. A key issue is to what extent the new technologies for PPI modulator R&D will enable this area to be commercially successful, and to meet the strategic needs of the industry for expanding the universe of targets for which drugs can be developed.

To see the report Advances in the Discovery of Protein-Protein Interaction Modulators, please click here.

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As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to  your company, please click here. We also welcome your comments on this or any other article on this blog.