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.

________________________________

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.

Amyloid precursor protein (APP)

As we mentioned in our August 19, 2012 article on Alzheimer’s disease (AD), the results of Phase 3 trials of Lilly’s amyloid-targeting monoclonal antibody (MAb) drug solanezumab, had been expected soon.

On August 24 2012, Lilly announced the top-line results of the two Phase 3, double-blind, placebo-controlled EXPEDITION trials of solanezumab in patients with mild-to-moderate Alzheimer’s disease. The primary endpoints, both cognitive and functional, were not met in either of these trials.

However, a pre-specified secondary analysis of pooled data across both trials showed statistically significant slowing of cognitive decline in the overall study population, and pre-specified secondary subgroup analyses of pooled data across both studies showed a statistically significant slowing of cognitive decline in patients with mild Alzheimer’s disease, but not in patients with moderate Alzheimer’s disease.

These results were reported in a press release.  What was absent was data from the trials. However, the Alzheimer’s Disease Cooperative Study (ADCS), (an academic national research consortium) will present its independent analysis of the data from the EXPEDITION studies at the American Neurological Association (ANA) meeting in Boston on October 8, 2012, and at the Clinical Trials on Alzheimer’s Disease (CTAD) meeting in Monte Carlo, Monaco, on October 30, 2012.

Once again, an amyloid pathway-targeting drug for Alzheimer’s disease that was taken into Phase 3 trials despite Phase 2 results that showed no statistically significant efficacy has failed in Phase 3. Solanezumab joins a list of such failed drugs that includes Myriad Pharmaceuticals’ Flurizan (tarenflurbil), Neurochem’s (now Bellus Health) Alzhemed (3-amino-1-propanesulfonic acid), and as of July 2012, Pfizer/Janssen’s bapineuzumab (“bapi”). Nevertheless, as in the Phase 2 results with bapi, Lilly sees hope for the drug in the results of secondary analyses.

On the day of the Lilly announcement, August 24 2012, Lilly executives and stock analysts turned the results of these trials into something “positive”, as the result of the secondary analysis. This resulted in a one-day 3.4 percent increase in the price of Lilly stock. However, the results of the secondary analysis do not give Lilly any basis for going to the FDA with a New Drug Application (NDA) for solanezumab. Nor do they provide any realistic hope for AD patients, the physicians who treat them, or caregivers of AD patients.

At best, Lilly’s secondary analysis gives rise to a hypothesis–that solanezumab–and presumably other anti-amyloid MAbs–will be effective in treating earlier-stage AD patients, especially those who have not suffered extensive, irreversible brain damage. This is the very same hypothesis that is now being tested by Roche/Genentech in its clinical trials of its anti-amyloid MAb crenezumab, as we discussed in our August 19, 2012 article. Genentech is testing its drug candidate in a Phase 2a trial in a very special population–members of a large Colombian kindred who harbor a mutation in presenilin 1 (PS1) that causes dominant early−onset familial AD.

A News Focus article in the 17 August 2012 issue of Science, written by science writer Greg Miller, PhD, discusses three upcoming clinical trials designed to test the “treat early-stage or presymptomatic AD with anti-amyloid MAbs” hypothesis. One of these studies is the Genentech trial of crenezumab in the extended family in Colombia.

Another of these studies is being conducted in conjunction with the Dominantly Inherited Alzheimer Network (DIAN), a consortium led by researchers at Washington University School of Medicine (St. Louis, MO). This study will include people with mutations in any of the three genes linked to early-stage, dominantly-inherited AD–PS1, PS2, and amyloid precursor protein (APP).

Initial studies, published ahead of print in the July 11 issue of the New England Journal of Medicine (NEJM) looked at changes in biomarkers and in cognitive ability as a function of expected age of AD onset in people with these mutations. Concentrations of amyloid-β1–42 (Aβ42) in the cerebrospinal fluid (CSF) appeared to decline 25 years before expected symptom onset. This decrease may reflect impaired clearance of Aβ42 from the brain, which may be a factor in the amyloid plaque increase that is associated with AD. Amyloid accumulation in the brain was detected 15 years before expected symptom onset. Other biomarkers, as well as cognitive impairment, were also followed in the study published in the NEJM. In the first stage of the actual trial, three drugs (which have not yet been selected) will be tested in this population, and changes in biomarkers and cognitive performance will be followed.

The third study, known as the Anti-Amyloid Treatment of Asymptomatic Alzheimer’s (A4) trial, will involve treating adults without mutations in any of the above three genes, whose brain scans show signs of amyloid accumulation. A4 is thus designed to study prevention of sporadic AD (by far the most common form of the disease). It will enroll 500 people age 70 or older who test positive on a scan of amyloid accumulation in the brain. (This is in contrast to the two trials in subjects with gene mutations, who are typically in their 30s or 40s.) A4 will also have a control arm of 500 amyloid-negative subjects. Amyloid-positive and control subjects will be entered into a three-year double-blind clinical trial that will look at changes in cognition with drug treatment. The A4 researchers [led by  Reisa Sperling, Brigham and Women’s Hospital/Harvard University (Boston, MA), and Paul Aisen, University of California, San Diego] plan to select a drug for testing by December 2012.

If Lilly wishes to test solanezumab in early-stage (or presymptomatic) sporadic AD, it will need to follow a similar methodology to the studies outlined in the new Science article, especially with respect to the use of biomarkers to define “early-stage” AD and to track the effects of the drug. Studies such as the DIAN biomarker study published in the NEJM used the positron emission tomography (PET) ligand Pittsburgh Compound-B (PiB-C11), to image amyloid plaques. However, the use of this compound is limited by the short half-life of carbon-11 (20.4 minutes). A new PET amyloid imaging agent, Amyvid (florbetapir F18 Injection) was developed by Lilly and approved by the FDA in April 2012. This compound contains fluorine-18, which has a half-life of 109.8 minutes. A recent study indicates that Amyvid provides comparable information to PiB-C11. If Lilly wishes to conduct new studies of solanezumab in early-stage or presymptomatic sporadic AD, it may wish to use Amyvid, as suggested in a comment to an August 24, 2012 solanezumab post in Derek Lowe’s blog “In the Pipeline”. However, the FDA, in its press release announcing the approval of Amyvid, warns that increased amyloid plaque content (as detected by Amyvid or Pittsburgh Compound-B) may be present in the brains of patients with non-AD neurologic conditions, and in older people with normal cognition. Thus defining or detecting “early-stage (or presymptomatic) sporadic AD” is difficult.

In any case, for Lilly to follow up on its secondary analyses of the Phase 3 clinical trials of solanezumab will necessitate additional long and expensive clinical trials, with no assurance of success. Lilly executives will need to determine if such a course is worth the risk, or whether it should invest in other R&D efforts that might have a higher probability of success.

__________________________________________

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 APP processing pathway

An exciting new study on Alzheimer’s disease (AD) was published in the 2 August issue of Nature. The study was carried out by researchers at deCode Genetics (Reykjavik Iceland) and their collaborators at Genentech and several academic institutions. A News and Views article by leading AD researcher Bart De Strooper and genomics researcher Thierry Voet (both at KU Leuven, Leuven, Belgium) analyzes this study and its implications.

Amyloid plaques are a central feature of AD.  They largely consist of amyloid-β (Aβ) peptides. Aβ peptides are formed via sequential proteolytic processing of the amyloid precursor protein (APP), catalyzed by two aspartyl protease enzymes–β-secretase and γ-secretase.  The β-site APP cleaving enzyme 1 (BACE1) cleaves APP predominantly at a unique site. However, γ-secretase cleaves the resulting carboxy-terminal fragment at several sites, with preference for positions 40 and 42. This leads to formation of amyloid-β1–40 (Aβ40) and Aβ1–42 (Aβ42) peptides. APP processing to yield Aβ peptides is illustrated by the figure at the top of this article.

By studying rare, familial cases of early-onset AD, human geneticists have identified three disease genes in these conditions— genes for APP, and for two presenilins, PS1 and PS2. The presenilins are components of γ-secretase, which exists as an intramembrane protease complex. Mainly because of these genetic studies, as well as studies in animal models and postmortem studies of AD brains, the majority of AD researchers have focused on the APP processing pathway and/or on aggregation of Aβ to form plaques as intervention points for therapeutic strategies. The hypothesis that this is the central AD disease pathway is called the “amyloid hypothesis”.

Up until the publication of the new deCode report, of the 30-odd coding mutations in APP that have been found, around 25 are pathogenic, usually resulting in autosomal dominant early-onset Alzheimer’s disease. Coding mutations at or near the β- or γ-proteolytic sites have appeared to result in overproduction of either total Aβ or a shift in the Aβ40:Aβ42 ratio towards formation of Aβ42, which is the more toxic of the two Aβ peptide. Until now, mutations in APP have not been implicated in the common, late-onset form of Alzheimer’s disease.

In the new deCode study, the researchers studied coding variants in APP in a set of whole-genome sequence data from 1,795 Icelanders. They identified a single nucleotide polymorphism (SNP), designated as rs63750847. The A allele of this SNP (rs63750847-A) results in an alanine to threonine substitution at position 673 in APP (A673T). The A673T mutation was found to be significantly more common in the elderly (age 85-100) control group (i.e., those without AD) than in the AD group. The researchers therefore concluded that the mutation is protective against AD.

The researchers also found that in a cohort of individuals over 80, those who were heterozygous for the A673T mutation performed better in a test of mental capacity than did control subjects. The authors concluded that the A673T mutation not only protects against AD, but also against the mild cognitive decline that is normally associated with old age.

In cellular studies (i.e., studies in cultured cells transfected with genes coding for wild type or mutant APP) and in biochemical studies, the researchers found that APP carrying the A673T mutation undergoes about 40% less cleavage by BACE1 than does wild-type APP, resulting in 40% less production of both Aβ40 and Aβ42.

The researchers conclude that the strong protective effect of the A673T mutation against AD provides proof of principle for the hypothesis that reducing the β-cleavage of APP (e.g., by use of BACE1 inhibitors, such as those being  developed by some pharmaceutical companies) may protect against the disease. (However, success in developing BACE1 inhibitors has been elusive.) Moreover, since the A673T allele also protects against cognitive decline in elderly individuals who do not have AD, AD and age-related mild cognitive decline may be mediated through the same or similar mechanisms.

Despite this compelling genetic finding, amyloid pathway-targeting drugs have not shown efficacy in Phase 3 trials

In our January 26, 2010 blog article, we discussed Phase 2 clinical trials of bapineuzumab, a monoclonal antibody (MAb) drug that is specific for Aβ, in mild to moderate AD. In that article, we referred to the drug as “Elan/Wyeth’s bapineuzumab”, after the original developers of the drug. As the result of mergers and acquisitions, the drug is now referred to as “Pfizer/Janssen’s bapineuzumab”. Many commentators call it “bapi” for short.

As we discussed in that article, the overall result of the Phase 2 trial was that there was no difference in cognitive function between patients in the bapi-treated and the placebo groups. However, the study did not have sufficient statistical power to exclude the possibility that there was such a difference. Retrospective analysis of the data from the trial suggested that bapi-treated patients who were not carriers of the apolipoprotein E epsilon4 allele (ApoE4) showed improved cognitive function as compared to placebo treatment. Given that this conclusion was reached via retrospective analysis, the idea that the bapi was efficacious in ApoE4 noncarriers was only a hypothesis, which would require prospective clinical trials to confirm. Janssen and Pfizer had been conducted large Phase 3 trials of bapi, which they prospectively segregated into ApoE4 carrier and noncarrier groups in order to test this hypothesis.

As of the past several weeks, the results of these Phase 3 trials have come in. On July 23rd, 2012, Pfizer announced the top-line results of an 18-month Janssen-led Phase 3 study of intravenous bapi in approximately 1,100 patients with mild to moderate Alzheimer’s disease who carry at least one ApoE4 allele. The drug failed to meet its co-primary endpoints (change in cognitive and functional performance compared to placebo) in that study. On August 6, 2012, Pfizer announced the top-line results of the corresponding Phase 3 study of intravenous bapi in patients with mild-to-moderate Alzheimer’s disease who do not carry the ApoE4 genotype. Once again, the co-primary clinical endpoints were not met. Based on these results, the companies decided to discontinue all other intravenous bapi studies in patients with mild-to-moderate Alzheimer’s disease.

The bapi development program continues a history of amyloid pathway-targeting drugs that were taken into Phase 3 trials despite Phase 2 results that showed no statistically significant efficacy. For example, we cited the cases of Myriad Pharmaceuticals’ Flurizan (tarenflurbil) and Neurochem’s (now Bellus Health) Alzhemed (3-amino-1-propanesulfonic acid) in our January 26, 2010 blog article.

Leading industry commentator Matthew Herper of Forbes referred to the failure of bapi as “the latest piece of evidence of the drug industry’s strange gambling problem.” Johnson & Johnson (the parent company of Janssen) spent more than $1 billion to invest in Elan and get one-quarter of bapi, and Wyeth (later Pfizer) and Elan put the drug into Phase 3, despite the Phase 2 failure of bapi.

The temptation for pharmaceutical companies to take a chance on an AD drug such as bapi, Flurizan, and Alzhemed is driven by the complete lack of disease-modifying AD drugs, and the thinking that even a not-very-effective drug that receives FDA approval might generate billions of dollars in annual sales. In the case of bapi there was also that tantalizing suggestion that bapi might show efficacy in the subset of patients who lacked ApoE4.

In an August 16, 2012 article in Forbes, Dr. John LaMattina (the former President of Pfizer Global R&D) engages in informed speculation as to why bapi was moved into Phase 3. Dr. LaMattina (in contrast to critics like Mr. Herper, who discounted the ApoE4 retrospective analysis as “data-dredging” that was “likely to be due to chance”) referred to the efficacy signal of the Phase 2 trials as “mixed” due to the ApoE4 analysis. He stated that such “mixed results” present an “agonizing” dilemma for a pharmaceutical company.

In deciding whether to go forward Phase 3 trials of bapi, Dr. LaMattina further speculates that the decision might have been influenced by stakeholders such as AD patient advocates, and scientists who strongly believed in the science behind bapi, especially the amyloid hypothesis. Moreover, bapi had been shown to be relatively safe. In addition, dropping bapi would have caused public relations damage. Dr. LaMattina concludes, based on this analysis, “…this was a situation where these companies were in possession of a relatively safe drug, with a modest chance of success in being efficacious in what may be the biggest scourge that society will face.  How can you not make this investment?” He reminds us that pharmaceutical R&D “is a high risk, high reward business”.

Nevertheless, bapi joined Flurizan and Alzhemed on the list of high-profile amyloid-pathway failures. Now a Phase 3 trial of Lilly’s solanezumab, another MAb drug that targets Aβ, is nearing completion, with the results expected in September. Published Phase 2 results were designed to test safety, not efficacy, and 12 weeks of drug treatment gave no change in cognitive function. Although the results of the Phase 3 trial will not be known until they are reported, analysts expect the drug to fail because of its similarity to bapi.

Why don’t amyloid pathway-targeting drugs show efficacy in clinical trials, despite the compelling genetic evidence for the amyloid hypothesis?

The almost standard answer to that question given by scientists and clinicians who support the amyloid hypothesis is that we have been testing the drugs too late in the course of AD progression, after the damage to the brain has become irreversible. Roche/Genentech is testing this idea in its clinical trials of its drug candidate crenezumab (licensed from AC Immune), which is yet another MAb drug that targets Aβ. In a 5-year Phase 2a clinical trial, Genentech is testing intravenous crenezumab in 300 cognitively healthy individuals from a large Colombian kindred who harbor the Glu280Ala (codon 280 Glu to Ala substitution) PS1 mutation. This mutation causes dominant early−onset familial AD, and is associated with increased levels of Aβ42 in plasma, skin fibroblasts, and the brain. Family members with this mutation begin showing cognitive impairment around age 45, and full dementia around age 51.

Genentech is conducting this trial in collaboration with the Banner Alzheimer’s Institute and the National Institutes of Health. The company says that this trial is the first-ever AD prevention study in cognitively healthy individuals. Genentech further says that the trial may help to determine if the amyloid hypothesis is correct–more specifically, it may help to determine if a drug that works by depleting amyloid plaques can be effective in preventing and/or treating AD.

Moreover, Genentech states that there is significant unmet medical need within this Colombian population. This large extended family may have as many as 5,000 living members, and no other population in the world offers a sufficiently large number of mutation carriers close to the age of potential disease onset for a study to determine whether a prevention treatment may work. This effort by Genentech thus represents an application of a rare disease strategy to AD.

It is also possible, however that drugs that work by lowering levels of Aβ will not be efficacious in treating AD, even if administered early in the disease process. This may be true despite the findings of the new genetic study by the deCode Genetics group. For example, in their Nature News and Views article, Drs. De Strooper and Voet remind us that if the A673T mutation indeed works via lowering of Aβ levels, it works via lifelong lowering of Aβ, not lowering of Aβ in patients who already have AD, as in all clinical trials so far of anti-Aβ antibodies. (Even Genentech’s Colombian trial may involve lowering of Aβ levels relatively late in the course of exposure of patients to a disease process that will result in AD.)

Moreover, as these authors speculate on the basis of work on another mutation at the same site in the APP protein, it is possible that the protective effect of the A673T mutation may be due to changing the aggregation properties of Aβ peptides, resulting in a less-toxic form of Aβ. If true, this would mean that the protective effect of the A673T mutation is due to qualitative, rather than quantitative changes in Aβ. In that case, the finding of protection from AD by the A673T mutation might not be as predictive of the efficacy of such Aβ-lowering treatments as the use of anti-Aβ MAb drugs as drug developers might like.
__________________________________________

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.

__________________________________________

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.

__________________________________________

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.