Posts Tagged ‘safety guidelines’

NIH Considers Guidelines for CAR-T therapy: Report from Recombinant DNA Advisory Committee

Reporter: Stephen J. Williams, Ph.D.

In the mid to late 1970’s a public debate (and related hysteria) had emerged surrounding two emerging advances in recombinant DNA technology;

  1. the development of vectors useful for cloning pieces of DNA (the first vector named pBR322) and
  2. the discovery of bacterial strains useful in propagating such vectors

As discussed by D. S, Fredrickson of NIH’s Dept. of Education and Welfare in his historical review” A HISTORY OF THE RECOMBINANT DNA GUIDELINES IN THE UNITED STATES” this international concern of the biological safety issues of this new molecular biology tool led the National Institute of Health to coordinate a committee (the NIH Recombinant DNA Advisory Committee) to develop guidelines for the ethical use, safe development, and safe handling of such vectors and host bacterium. The first conversations started in 1974 and, by 1978, initial guidelines had been developed. In fact, as Dr. Fredrickson notes, public relief was voiced even by religious organizations (who had the greatest ethical concerns)

On December 16, 1978, a telegram purporting to be from the Vatican was hand delivered to the office of Joseph A. Califano, Jr., Secretary of Health, Education,

and Welfare. “Habemus regimen recombinatum,” it proclaimed, in celebration of the

end of a long struggle to revise the NIH Guidelines for Research Involving

Recombinant DNA Molecules

The overall Committee resulted in guidelines (2013 version) which assured the worldwide community that

  • organisms used in such procedures would have limited pathogenicity in humans
  • vectors would be developed in a manner which would eliminate their ability to replicate in humans and have defined antibiotic sensitivity

So great was the success and acceptance of this committee and guidelines, the NIH felt the Recombinant DNA Advisory Committee should meet regularly to discuss and develop ethical guidelines and clinical regulations concerning DNA-based therapeutics and technologies.

A PowerPoint Slideshow: Introduction to NIH OBA and the History of Recombinant DNA Oversight can be viewed at the following link:

Please see the following link for a video discussion between Dr. Paul Berg, who pioneered DNA recombinant technology, and Dr. James Watson (Commemorating 50 Years of DNA Science):

The Recombinant DNA Advisory Committee has met numerous times to discuss new DNA-based technologies and their biosafety and clinical implication including:

A recent Symposium was held in the summer of 2010 to discuss ethical and safety concerns and discuss potential clinical guidelines for use of an emerging immunotherapy technology, the Chimeric Antigen Receptor T-Cells (CART), which at that time had just been started to be used in clinical trials.

Considerations for the Clinical Application of Chimeric Antigen Receptor T Cells: Observations from a Recombinant DNA Advisory Committee Symposium Held June 15, 2010[1]

Contributors to the Symposium discussing opinions regarding CAR-T protocol design included some of the prominent members in the field including:

Drs. Hildegund C.J. Ertl, John Zaia, Steven A. Rosenberg, Carl H. June, Gianpietro Dotti, Jeffrey Kahn, Laurence J. N. Cooper, Jacqueline Corrigan-Curay, And Scott E. Strome.

The discussions from the Symposium, reported in Cancer Research[1]. were presented in three parts:

  1. Summary of the Evolution of the CAR therapy
  2. Points for Future Consideration including adverse event reporting
  3. Considerations for Design and Implementation of Trials including mitigating toxicities and risks

1. Evolution of Chimeric Antigen Receptors

Early evidence had suggested that adoptive transfer of tumor-infiltrating lymphocytes, after depletion of circulating lymphocytes, could result in a clinical response in some tumor patients however developments showed autologous T-cells (obtained from same patient) could be engineered to express tumor-associated antigens (TAA) and replace the TILS in the clinical setting.

However there were some problems noticed.

  • Problem: HLA restriction of T-cells. Solution: genetically engineer T-cells to redirect T-cell specificity to surface TAAs
  • Problem: 1st generation vectors designed to engineer T-cells to recognize surface epitopes but engineered cells had limited survival in patients.   Solution: development of 2nd generation vectors with co-stimulatory molecules such as CD28, CD19 to improve survival and proliferation in patients

A summary table of limitations of the two types of genetically-modified T-cell therapies were given and given (in modified form) below

                                                                                                Type of Gene-modified T-Cell

Limitations aβ TCR CAR
Affected by loss or decrease of HLA on tumor cells yes no
Affected by altered tumor cell antigen processing? yes no
Need to have defined tumor target antigen? no yes
Vector recombination with endogenous TCR yes no

A brief history of construction of 2nd and 3rd generation CAR-T cells given by


Differences between  second- and third-generation chimeric antigen receptor T cells. (Adapted by permission from the American Association for Cancer Research: Lee, DW et al. The Future Is Now: Chimeric Antigen Receptors as New Targeted Therapies for Childhood Cancer. Clin Cancer Res; 2012;18(10); 2780–90. doi:10.1158/1078-0432.CCR-11-1920)

Constructing a CAR T Cell (from

The first efforts to engineer T cells to be used as a cancer treatment began in the early 1990s. Since then, researchers have learned how to produce T cells that express chimeric antigen receptors (CARs) that recognize specific targets on cancer cells.

The T cells are genetically modified to produce these receptors. To do this, researchers use viral vectors that are stripped of their ability to cause illness but that retain the capacity to integrate into cells’ DNA to deliver the genetic material needed to produce the T-cell receptors.

The second- and third-generation CARs typically consist of a piece of monoclonal antibody, called a single-chain variable fragment (scFv), that resides on the outside of the T-cell membrane and is linked to stimulatory molecules (Co-stim 1 and Co-stim 2) inside the T cell. The scFv portion guides the cell to its target antigen. Once the T cell binds to its target antigen, the stimulatory molecules provide the necessary signals for the T cell to become fully active. In this fully active state, the T cells can more effectively proliferate and attack cancer cells.

2. Adverse Event Reporting and Protocol Considerations

The symposium had been organized mainly in response to two reported deaths of patients enrolled in a CART trial, so that clinical investigators could discuss and formulate best practices for the proper conduct and analysis of such trials. One issue raised was lack of pharmacovigilence procedures (adverse event reporting). Although no pharmacovigilence procedures (either intra or inter-institutional) were devised from meeting proceedings, it was stressed that each institution should address this issue as well as better clinical outcome reporting.

Case Report of a Serious Adverse Event Following the Administration of T Cells Transduced With a Chimeric Antigen Receptor Recognizing ERBB2[2] had reported the death of a patient on trial.

In A phase I clinical trial of adoptive transfer of folate receptor-alpha redirected autologous T cells for recurrent ovarian cancer[3] authors: Lana E Kandalaft*, Daniel J Powell and George Coukos from University of Pennsylvania recorded adverse events in pilot studies using a CART modified to recognize the folate receptor, so it appears any adverse event reporting system is at the discretion of the primary investigator.

Other protocol considerations suggested by the symposium attendants included:

  • Plan for translational clinical lab for routine blood analysis
  • Subject screening for pulmonary and cardiac events
  • Determine possibility of insertional mutagenesis
  • Informed consent
  • Analysis of non T and T-cell subsets, e.g. natural killer cells and CD*8 cells

3. Consideration for Design of Trials and Mitigating Toxicities

  • Early Toxic effectsCytokine Release Syndrome– The effectiveness of CART therapy has been manifested by release of high levels of cytokines resulting in fever and inflammatory sequelae. One such cytokine, interleukin 6, has been attributed to this side effect and investigators have successfully used an IL6 receptor antagonist, tocilizumab (Acterma™), to alleviate symptoms of cytokine release syndrome (see review Adoptive T-cell therapy: adverse events and safety switches by Siok-Keen Tey).


Below is a video form Dr. Renier Brentjens, M.D., Ph.D. for Memorial Sloan Kettering concerning the finding he made that the adverse event from cytokine release syndrome may be a function of the tumor cell load, and if they treat the patient with CAR-T right after salvage chemotherapy the adverse events are alleviated..

Please see video below:

http link:

  • Early Toxic effects – Over-activation of CAR T-cells; mitigation by dose escalation strategy (as authors in reference [3] proposed). Most trials give billions of genetically modified cells to a patient.
  • Late Toxic Effectslong-term depletion of B-cells . For example CART directing against CD19 or CD20 on B cells may deplete the normal population of CD19 or CD20 B-cells over time; possibly managed by IgG supplementation

 Please look for a Followup Post concerning “Developing a Pharmacovigilence Framework for Engineered T-Cell Therapies”


  1. Ertl HC, Zaia J, Rosenberg SA, June CH, Dotti G, Kahn J, Cooper LJ, Corrigan-Curay J, Strome SE: Considerations for the clinical application of chimeric antigen receptor T cells: observations from a recombinant DNA Advisory Committee Symposium held June 15, 2010. Cancer research 2011, 71(9):3175-3181.
  2. Morgan RA, Yang JC, Kitano M, Dudley ME, Laurencot CM, Rosenberg SA: Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Molecular therapy : the journal of the American Society of Gene Therapy 2010, 18(4):843-851.
  3. Kandalaft LE, Powell DJ, Jr., Coukos G: A phase I clinical trial of adoptive transfer of folate receptor-alpha redirected autologous T cells for recurrent ovarian cancer. Journal of translational medicine 2012, 10:157.

Other posts on this site on Immunotherapy and Cancer include

Report on Cancer Immunotherapy Market & Clinical Pipeline Insight

New Immunotherapy Could Fight a Range of Cancers

Combined anti-CTLA4 and anti-PD1 immunotherapy shows promising results against advanced melanoma

Molecular Profiling in Cancer Immunotherapy: Debraj GuhaThakurta, PhD

Pancreatic Cancer: Genetics, Genomics and Immunotherapy

$20 million Novartis deal with ‘University of Pennsylvania’ to develop Ultra-Personalized Cancer Immunotherapy

Upcoming Meetings on Cancer Immunogenetics

Tang Prize for 2014: Immunity and Cancer

ipilimumab, a Drug that blocks CTLA-4 Freeing T cells to Attack Tumors @DM Anderson Cancer Center

Juno’s approach eradicated cancer cells in 10 of 12 leukemia patients, indicating potential to transform the standard of care in oncology


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A New Therapy for Melanoma

Reporter  Larry H Bernstein, MD

S Andrews, R Holden.  Characteristics and management of immune-related adverse effects associated with ipilimumab, a new immunotherapy for metastatic melanoma. Cancer Management and Research (Dovepress: open access) 11 Sept, 2012; 4:299-307.

This report is an immediate followup to a post on Ulcerative Colitis, a chronic inflammatory condition of the colonic mucosa that is now ready for phase 3 clinical trial with a breakthrough drug that blocks the action of “T-cell lymphokine” activity of IL-2, 4, 7, 15 and 21.  This also deals with a breakthrough immunotherapy for a common skin malignancy, melanoma, the sixth most common cancer (bearing no ontogenetic resemblance to the GI disease), that accounted for 8790 in the US in 2011. Approximately 84% of cases present with local disease (stages I and II), 8% of patients have regional disease, and only 4% show distant metastases, with 4% being unstaged. Melanomas characteristically occur after puberty.    Lesions that are by all means identical to melanoma behave as a benign pigmented mole in a child.  The melanoma is a raised pigmented lesion that when it invades deeply through the dermal layer and metastasizes, would be identify this late stage for treatment of the disease.  Stage IV disease is defined as distant skin involvement, soft tissue involvement, lung, and/or visceral sites.  Survival rates are highly dependent on the stage of metastatic disease,  with stage IV melanoma patients showing 15% survival rate at 5 years, but the survival rate drops to 3% at 5 years with brain metastasis. The therapeutic options for extensive disease or for metastatic disease have been until recently limited to entry into a clinical trial, treatment with dacarbazine or high-dose interleukin-2 was the only therapy approved by the US Food and Drug Administration (FDA) with no survival benefit and greater toxicity of combination sequential therapy of dacarbazine + other  drugs.

Vemurafenib (Zelboraf®, Genentech, South San Francisco, CA) is a BRAF inhibitor that has demonstrated activity in patients with metastatic melanoma who harbor the V600E BRAF mutation. Recent interim results of a Phase III study have reported 6-month overall survival of 84% (95% confidence interval 78–89) in patients receiving vemurafenib compared with 65% in the dacarbazine arm (95% confidence interval 56–73) of the study.14 The rate of progression-free survival was also improved in the vemurafenib arm, and this agent has received approval from the FDA for the treatment of patients with unresectable or metastatic melanoma whose tumors harbor the V600E mutation.  However, the long-term efficacy and safety of vemurafenib has yet to be determined.  Introduce Ipilimumab (Bristol Myers Squibb), the first drug approved for the treatment of melanoma by the FDA which has shown a survival benefit in a randomized Phase III study.

Mechanism of Action:  Ipilimumab is a monoclonal antibody that blocks cytotoxic T lymphocyte antigen-4, an inhibitor of T cell activation, thereby potentiating an immune response.  It’s unique mechanism of action differentiates it from chemotherapies, in that it targets the immune system rather than directly targeting the tumor itself.  Recall that in the previous GI inflammatory case the target was T-lymphocytes 2, 4, 7, 15, and 21. So this treatment is more narrowly focused.

Most recent Pase III Trial Result:  A Phase III study of ipilimumab in treatment-naïve patients with unresectable stage III or IV melanoma was recently published.  This study was done using a higher, experimental dose of ipilimumab at 10 mg/kg in combination with dacarbazine compared with dacarbazine alone. The combination arm of the study showed significantly higher survival rates in patients who received dacarbazine alone at one year (47.3% versus 36.3%), 2 years (28.5% versus 17.9%), and 3 years (20.8% versus 12.2%, hazard ratio for death 0.72; P , 0.001). The study confirmed the overall survival benefit of ipilimumab and it demonstrated an acceptable safety profile.

Summary of pharmaceutical treatment options

• Dacarbazine has low toxicity, is well tolerated, minimally effective, and has limited progression-free survival

• High-dose interleukin-2 has high toxicity, is used in highly selected patients, and has limited efficacy

• Combination biochemotherapeutic regimens have not been shown to have an overall survival benefit, and have added toxicity

• Vemurafenib shows a rapid response, has improved 6-month overall survival and progression-free survival, but lacks durability

• Ipilimumab has a unique side effect profile, and has been shown to improve one-year and two-year overall survival.

Immune-related AEs with ipilimumab

The most common safety events associated with ipilimumab therapy are immune-related; a recent pooled analysis of 14 completed Phase I–III ipilimumab clinical trials showed that 64.2% of patients experienced an immune-related adverse event (AE) of any grade.20 Immune-related AEs are likely reflective of the immune-based mechanism of action of ipilimumab and may affect various organs. An overview of the rate of immune-related AEs associated with ipilimumab 3 mg/kg in the Phase III MDX010-20 registration trial is shown in Table 1 (Rates of immune-related adverse events from the MDX010-20 registration trial, which included previously treated patients with unresectable stage III or IV melanoma treated with ipilimumab 3 mg/kg alone, a control vaccine alone (glycoprotein 100), or a combination of both ipilimumab and glycoprotein 100).  The most common immune-related AEs included toxicities of the skin, gastrointestinal tract, endocrine system, and liver.  The majority of immune-related AEs initially manifest during induction phase; however, a minority occurs weeks to months after discontinuation of ipilimumab. Time to resolution of immune-related AEs experienced by patients varied from 4.3 to 7.7 weeks on average across all studies.

Zelboraf® (vemurafenib) package insert. South San Francisco, CA: Genentech USA Inc,; 2011.
Hodi FS, O’Day SJ, McDermott DF, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med. 2010; 363:711–723.

Patterns of response with ipilimumab

In addition to the safety events, which may be tied to the unique mechanism of action of ipilimumab, unique clinical responses might also be reflective of the immune-related mechanism of action. (Table 2) Response patterns with ipilimumab include two standard responses that are commonly observed with other cytotoxic therapies, the first being an immediate decline in overall tumor burden with no new lesions and the second being stable disease. Stable disease in some patients on ipilimumab can be followed by a slow and steady decline in tumor burden.  However, throughout clinical development of ipilimumab, two additional, novel response patterns have been observed in patients, while still being shown to be associated with improved survival in patients. The first is a reduction in overall tumor burden in the presence of new lesions and the second is an initial increase followed by a steady decrease in tumor volume.

Patterns of response with ipilimumab

• Immediate response in baseline lesions, without the presence of new lesions

• Durable stable disease (SD), which may be followed by a slow, steady decline in total tumor burden

• Response after an increase in total tumor burden

• Response in presence of new lesions (which may have been present at baseline but were radiographically undetectable)

Notes: All patterns of response have been associated with response in patients and to improved survival.

Hoos A, Ibrahim R, Korman A, et al. Development of ipilimumab: contribution to a new paradigm for cancer immunotherapy. Semin Oncol. 2010;37(5):533–546.

Ibrahim R, Berman D, de Pril V, et al. Ipilimumab safety profile: summary of findings from completed trials in advanced melanoma. J Clin Oncol. 2011;29 Suppl:Abstract 8583.

Wolchok JD, Hoos A, O’Day S, et al. Guidelines for the evaluation of immune therapy activity in solid tumors: immune-related response criteria. Clin Cancer Res. 2009;15:7412–7420.

Hoos A, Eggermont AM, Janetzki S, et al. Improved endpoints for cancer immunotherapy trials. J Natl Cancer Inst. 2010;102:1388–1397.


Ipilimumab is a novel immunotherapeutic agent approved by the FDA for unresectable and metastatic melanoma. Due to its characteristic and distinctive mechanism of action, ipilimumab elicits a number of specific immune-related AEs. Time to onset and resolution of ipilimumab-associated immune-related AEs follow a predictable temporal pattern but can vary from patient to patient.

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