Steven A Rosenberg MD and the Race for CAR T-Cell Cancer Therapy

This article is part one
For part two, Click Here

by Jeffrey Dach MD

Expecting the Unexpected

Laboring tediously for hours on end, medical students inject cancer cells into mice, producing many publications in prestigious journals.  As you might expect, all the cancer injected mice died promptly from cancer.  All except one.  The unexpected happened.  This one mouse survived, and was renamed, “the mouse that killed cancer.”  As Luis Pasteur once said, Fortune favors the prepared mind.  Above Left Image Steven A Rosenberg MD PhD courtesy of OncLive.

Spontaneous Regression of Cancer in the Mouse

Once identified as a “cancer killing” mouse, the little furry fellow was earmarked for study. These were exciting times, and there were many questions.  Why didn’t this mouse die of cancer like all the others?   How was this mouse able to defeat the injected cancer cells?   What kind of  immune system protected this mouse? Left Image: Laboratory Mouse courtesy of wikimedia commons.

A Mouse Immune to Cancer

Over the next 3 years, studies showed this strain of mice had an innate immunity to cancer, a genetic trait passed on to the offspring.  Their T-Cells, T-lymphocytes, recognize and kill cancer cells, just as if cancer was an invading microorganism.  These mice were called SR/CR mice, which stands  for Spontaneous Regression/Complete Resistant to Cancer.(2)

Saving Other Mice From Cancer

What about the other plain mice with no immunity to cancer who quickly succumb to injected cancer cells?   Could these plain mice be protected from cancer by transferring the immune system from a SR/CR mouse?  What if we infused the T-Cells, the immune cells from a SR/CR mouse into a plain mouse?

More experiments showed yes, this is correct.  Protection from the injected cancer cells could be transferred to plain mice after transfusion with T-Cells from SR/CR mice.(3) In addition, the protective SR/CR T-Cells still retained activity after many weeks of cold storage.(4)

Human Mice – Spontaneous Regression of Cancer

What about us humans? Do we have similar immunity to cancer? Are some of us humans “immune” from cancer?  The answer is “Yes”, and this is called spontaneous remission of cancer, reported many times in the medical literature.  Spontaneous remission/regression has been reported for neuroblastoma, renal cell carcinoma, malignant melanoma and lymphomas/leukemias (see Papac RJ and Chodorowski Z). (5)(6)

The Legend of Sir William Osler – Spontaneous Regression

In 1901, the legendary Dr William Osler, reported spontaneous remission in fourteen cases of breast cancer .  (reference: The Medical Aspects of Carcinoma of the Breast,  Spontaneous Disappearance of Secondary Growths OSLER W., American Medicine: April 6 1901; 17-19; 63-66.)

Spontaneous Regression in Breast Cancer

A study by Dr Gilbert Welch concluded that small breast cancers may spontaneously regress as reported by  Gina Kolata in the New York Times .  Spontaneous regression in the cancer patient is a victory of the immune system over the malignant potential of the cancer mass.  Left Image Sir William Osler 1912 courtesy of wikimedia.

Adoptive Immunotherapy – Dr Steven A Rosenberg NIH

Inspired by this SR/CR Immune Mouse in which immunity to cancer may be transferred from one mouse to another,  Steven A Rosenberg MD at the National Cancer Institute, developed a cancer treatment using T-Cells infused into cancer patients.   He called this “Adoptive Cell Transfer Immunotherapy”, and  results have been remarkable.(7)  

Dr Rosenberg ‘s treatment involves pre-treating the patient’s T lymphocytes to increase the anti-tumor activity.  The patient’s blood is drawn, the lymphocytes isolated, and then activated and cloned in a test tube.  These activated lymphocytes are then re-infused into the patient. This method is very effective for metastatic melanoma, producing tumor regression in 50% of patients in clinical trials.  See Rosenberg’s case images showing tumor regression (7). See Figure 2 from his article showing examples of tumor regression in patients receiving immune cells, lymphocytes (white cells).(7)

Enter the CAR T Cell, Chimeric Antigen Receptor Engineered T-Cell

What if we could re-engineer the patients T-Cells, the immune cells,  to recognize and attack surface antigens on the cancer cell?  Using modern biotechnology techniques this can be done.  The technique is called CAR, which stands for Chimeric Antigen Receptor.(8-12)  See left diagram of CAR T-Cell courtesy of The Pharmaceutical Journal .

CD19 Marker on B-Cells

The most promising efforts using CAR T Cells have been directed at  the CD19 marker on B-Cell malignancies such as Leukemia and Lymphoma.  These  B cell cancers have the CD19 surface antigen marker allowing the CAR T-Cells to recognize and destroy them.(8-12)  

As of 2014,  there have been 13 publications of clinical trials using CAR T cells for  treatment of B-cell malignancies targeting the CD19 or CD20 surface antigen.  These have, showing dramatic remissions in a number of patients with refractory B-cell lymphoma. (10)(12)  The CAR technique has also been tried in neuroblastoma, and sarcoma patients. (11)

Low Dose Chemotherapy Followed by CAR

Dr Steven Rosenberg co-authored an abstract at the June 2016 ASCO meeting, presenting  22 patients with B cell malignancies treated with low dose chemotherapy followed by anti-CD19 CAR engineered T-Cells.(13)  Most of these cases were refractory to chemotherapy or had relapsed after autologous stem cell transplant (these are stem cells harvested from the patient and re-infused).  Following treatment, about half of the patients had ongoing complete remissions, an impressive result.(13)

CAR added to Allogeneic Transplant Protocol

Dr Steven Rosenberg and Jennifer Brudno reported in 2016 using anti-CD19 engineered T cells as part of an allogeneic transplant protocol.(14)  Twenty lymphoma patients with progressive disease after allogeneic stem cell transplant were given anti CD-19 T-Cells prepared from the transplant donor.  Results were favorable with the most impressive remissions in ALL (acute lymphoblastic leukemia).  There was no graft-versus-host disease and toxicities were acceptable.   The authors predicted that in the near future, CAR engineered T Cells will be routinely added to allogeneic stem cell transplant protocols. (14,15)

The Race for CAR-T Cell Approval

Three biotech companies, Juno, Kite and Novartis, are in clinical trials racing towards FDA approval for their CAR T-Call product lines.  The treatment of B cell cancers has been successful because B Cells are the only ones expressing CD19 marker, and also because we can live without B cells.  The first CAR T-cell therapy for B-Cell cancers could enter the market in 2017.(17)  Left Image foot race courtesy NY Times.

According to Rachel Webster: “sInce 2014, CAR T-cell therapies have achieved impressive complete response (CR) rates of up to 90% in pediatric and adult patients with relapsed or refractory (R/R) acute lymphocytic leukemia (ALL) and non-Hodgkin’s lymphoma (NHL); durable remissions and six-month survival of more than 70% have been reported.” (17)

Making CAR T-Cell Therapy More Effective: Ibrutinib

Results of CAR T Cell Therapy for Non-Hodgins Lymphoma can be improved with co-administration of Ibrutinib, (Brutons Tyrosine Kinase Inhibotor).  Studies by Steven Schuster at U Penn using mouse – mantle cell lymphoma xenografts show more durable long term remission when ibrutinib is co-adminsisterd with the CAR T cell therapy. (28)

According to Dr Wasik in a 2015 report, Mantle Cell Lymphoma cells have CB1 Cannabinoid receptors which are not present on normal lymphocytes, and found in the central nervous system, perhaps explaining ready attachment to nervous tissue.  For prevention or treatment of nervous system lymphoma, Ibrutinib is an excellent choice, since it readily crosses the blood brain barrier, and showed impressive results in three cases mantle cell lymphoma with CNS relapse reported in 2015 by Dr Sophie Bernard.in Paris.(32)  “All three patients had dramatic and rapid responses with almost immediate recovery from CNS symptoms. “(32)

Cytokine Release Syndrome

An adverse side effect of CAR T-Cell therapy is cytokine release syndrome (CRS), the release of inflammatory cytokines upon interaction of the CAR T cells with the malignant cells.  This adverse effect seems most pronounced in cases with heavy disease burden, highlighting the need for cytoreductive chemotherapy prior to T Cell infusion.  “Severe (and potentially fatal) CRS has been observed in approximately one-third of R/R ALL patients.”(17)  Another potential adverse effect is long lasting loss of the B-cell population which puts the patient at risk for infections. (16)

A Treatment with Rock-Star Status Suffers SetBack

In July 2016, the FDA temporarily halted Juno’s CAR-T clinical trials at Memorial Sloan Kettering Cancer Center in New York after three patient deaths due to cerebral edema from cytokine-release syndrome (CRS),(18)  They quickly resumed enrollment after a short one week delay.

Wounded Juno Allows KITE to FLY

As Juno falls behind, KITE captures the lead with their KTE-619 CAR T-Cell product.  In June 2016,  Kite opened their 43,000 square foot manufacturing facility for commercial production of KTE-619 CAR-T Cells, with capacity to treat 5,000 patients per year.  The new plant will be operational by Dec 2016    Turnaround time from lymphopheresis to reinfusion of the KTE C19 product is approximately 2 weeks, one of the fastest in the industry.(19)  Shawn Tomasello of KITE says he is  “building out a commercial team that will target 50 to 70 cancer centers.” (20)

Conclusion: One can only stand in awe of Dr Steven A Rosenberg and his almost herculean accomplishments in the field of cancer immunotherapy  ultimately bearing fruit as CAR T-cell Therapy for B-Cell Malignancies.   Of the three biotech companies now in a race for FDA approval, KITE seems poised to grasp the prize.

This article is part one, for part TWO, Click Here

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Jeffrey Dach MD
7450 Griffin Road Suite 190
Davie, Fl 33314
954-792-4663

Links and References

(1) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC164507/
Proc Natl Acad Sci U S A. 2003 May 27; 100(11): 6682–6687.
Spontaneous regression of advanced cancer: Identification of a unique genetically determined, age-dependent trait in mice. Zheng Cui, Mark C. Willingham, Amy M. Hicks, Martha A. Alexander-Miller et al.

We have established and studied a colony of mice with a unique trait of host resistance to both ascites and solid cancers induced by transplantable cells. One dramatic manifestation of this trait is age-dependent spontaneous regression of advanced cancers. This powerful resistance segregates as a single-locus dominant trait, is independent of tumor burden, and is effective against cell lines from multiple types of cancer. During spontaneous regression or immediately after exposure, cancer cells provoke a massive infiltration of host leukocytes, which form aggregates and rosettes with tumor cells. The cytolytic destruction of cancer cells by innate leukocytes is rapid and specific without apparent damage to normal cells. The mice are healthy and cancer-free and have a normal life span. These observations suggest a previously unrecognized mechanism of immune surveillance, which may have potential for therapy or prevention of cancer.

(2) http://www.cancerimmunity.org/v6p11/060911.htm
Cancer Immunity, Vol. 6, p. 11 (31 October 2006) Submitted: 28 March 2006. Resubmitted: 17 July 2006. Accepted: 28 September 2006. Effector mechanisms of the anti-cancer immune responses of macrophages in SR/CR mice.

Amy M. Hicks et al. The killing of cancer cells in SR/CR mice requires three distinct phases. First, the leukocytes must migrate to the site of cancer cells after sensing their presence. Second, they must recognize the unique properties of the cancer cell surface and make tight contact with it. Third, the effector mechanisms must finally be delivered to target cells. The difference between SR/CR and WT mice seems to lie in one of the first two phases. Upon challenge with cancer cells, WT mice lack leukocyte infiltration and rosette formation. Apparently, the mutation in SR/CR mice renders the leukocytes capable of sensing unique diffusible and surface signals from cancer cells, and of responding to the activation signals by migration and physical contact. Once the first two phases are accomplished, unleashing the pre-existing effector mechanisms for killing seems to ensue by default. Therefore, the mutated gene (or genes) likely determines whether leukocytes interpret the signals from cancer cells as inhibition, as in WT leukocytes, or as activation of migration and target recognition, as in SR/CR leukocytes. Identifying the mutated gene (or genes) will likely explain this unique resistance to cancer through immunity.

(3) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1458507/?tool=pubmed
Proc Natl Acad Sci U S A. 2006 May 16; 103(20): 7753–7758. Immunology Transferable anticancer innate immunity in spontaneous regression/complete resistance mice. Amy M. Hicks et al.

(4) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2749872/?tool=pubmed
BMC Cancer. 2009; 9: 328.

Impact of sex, MHC, and age of recipients on the therapeutic effect of transferred leukocytes from cancer-resistant SR/CR mice
John R Stehle, Jr,1 Michael J Blanks,2 Gregory Riedlinger,1,3 Jung W Kim-Shapiro,1 Anne M Sanders,1 Jonathan M Adams,1 Mark C Willingham,1 and Zheng Cui1
1Department of Pathology, Wake Forest University School of Medicine Winston-Salem, North Carolina 27157, USA

Abstract Background
Spontaneous Regression/Complete Resistant (SR/CR) mice are resistant to cancer through a mechanism that is mediated entirely by leukocytes of innate immunity. Transfer of leukocytes from SR/CR mice can confer cancer resistance in wild-type (WT) recipients in both preventative and therapeutic settings. In the current studies, we investigated factors that may impact the efficacy and functionality of SR/CR donor leukocytes in recipients.

Spontaneous regression of cancer in Humans

(5)http://www.ncbi.nlm.nih.gov/pubmed/9891219
In Vivo. 1998 Nov-Dec;12(6):571-8.
Spontaneous regression of cancer: possible mechanisms. Papac RJ.
Section of Medical Oncology, Yale University School of Medicine, New Haven, CT 06520, USA.

Spontaneous regression of cancer is reported in virtually all types of human cancer, although the greatest number of cases are reported in patients with neuroblastoma, renal cell carcinoma, malignant melanoma and lymhomas/leukemias. Study of patients with these diseases has provided most of the data regarding mechanisms of spontaneous regression. Mechanisms proposed for spontaneous regression of human cancer include: immune mediation, tumor inhibition by growth factors and/or cytokines, induction of differentiation, hormonal mediation, elimination of a carcinogen, tumor necrosis and/or angiogenesis inhibition, psychologic factors, apoptosis and epigenetic mechanisms. Clinical observations and laboratory studies support these concepts to a variable extent. The induction of spontaneous regression may involve multiple mechanisms in some cases although the end result is likely to be either differentiation or cell death. Elucidation of the process of spontaneous regression offers the possibility of improved methods of treating and preventing cancer.

(6) http://www.ncbi.nlm.nih.gov/pubmed/17724923
Przegl Lek. 2007;64(4-5):380-2.
[Spontaneous regression of cancer–review of cases from 1988 to 2006] Chodorowski Z, Anand JS, Wiśniewski M, Madaliński M, Wierzba K, Wiśniewski J.  Katedra i Klinika Chorób Wewnetrznych, Geriatrii i Toksykologii Klinicznej, Akademii Medycznej w Gdańsku.

Spontaneous regression of malignant tumours is a rare and enigmatic phenomenon. We reviewed the cases of spontaneous regression of cancer in medical literature according to MEDLINE database in the period 1988-2006 and compared them with similar reviews from 1900-1987 period. The number of reported cases of spontaneous regression increased steadily in XX century, probably due to a rising interest in this problem and new possibilities of radiological and biopsy examinations. Spontaneous regression of malignancy was reported in almost all types of human cancer, although the greatest number of cases in years 1988-2006 were reported in patients with nephroblastoma, renal cell carcinoma, malignant melanoma, lymphoma. Elucidation of the process of spontaneous regression offers the possibility of improved methods of preventing andlor treating cancer.

Adoptive Cell Therapy ACT

(7) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2553205/?tool=pubmed
Nat Rev Cancer. 2008 April; 8(4): 299–308.
Adoptive cell transfer: a clinical path to effective cancer immunotherapy
Steven A. Rosenberg, Nicholas P. Restifo, James C. Yang, Richard A. Morgan, and Mark E. Dudley. Surgery Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 10 Center Drive, Bethesda, Maryland 20892, USA.

Adoptive cell therapy (ACT) using autologous tumour-infiltrating lymphocytes has emerged as the most effective treatment for patients with metastatic melanoma and can mediate objective cancer regression in approximately 50% of patients. The use of donor lymphocytes for ACT is an effective treatment for immunosuppressed patients who develop post-transplant lymphomas. The ability to genetically engineer human lymphocytes and use them to mediate cancer regression in patients, which has recently been demonstrated, has opened possibilities for the extension of ACT immunotherapy to patients with a wide variety of cancer types and is a promising new approach to cancer treatment.

Figure 2
Examples of objective tumour regressions in patients receiving adoptive cell transfer of autologous anti-tumour lymphocytes following a lymphodepleting preparative regimen
In each case the pretreatment scans and photos are shown on the left and the post-treatment on the right. a | A 45-year-old male with metastatic melanoma to the liver (upper) and right adrenal gland (middle) who was refractory to prior treatment with high dose α interferon as well as high-dose interleukin 2 (IL2). He underwent a rapid regression of metastases and developed vitiligo (lower). b | A 55-year-old male with rapid tumour growth in the axilla as well as multiple brain metastases from metastatic melanoma that was refractory to prior treatment with high dose IL2 who underwent rapid regression of nodal and brain metastases.

The future of ACT

In contrast to common epithelial cancers, melanoma appears to be a tumour that naturally gives rise to anti-tumour T cells. However, other cancers are equally susceptible as the targets of reactive T cells. The susceptibility of melanoma to ACT provides optimism for the application of ACT to common epithelial cancers using TCR gene-modified lymphocytes.

A major problem with the application of ACT is that it is a highly personalized treatment and does not easily fit into current modes of oncological practice. The treatment is labour-intensive and requires laboratory expertise. In essence, a new reagent is created for each patient and this patient-specific nature of the treatment makes it difficult to commercialize. Pharmaceutical and biotechnology companies seek off-the-shelf drugs, easy to produce, vial and administer. From a regulatory standpoint, ACT might be more appropriately delivered as a service rather than as a ‘drug’. Blood banks have been instrumental in providing CD34+ haematopoietic stem cells for clinical studies and might be the ideal location for the generation of the anti-tumour T cells needed for ACT.

As modern science increasingly provides the physician with sophisticated information about the unique aspects of an individual cancer, changes in the modes of care delivery need to accommodate this. The ability to use this patient-specific information can lead to a new era of personalized medicine in which individual treatments, such as ACT, are devised for each patient.Studies of ACT have clearly demonstrated that the administration of highly avid anti-tumour T cells directed against a suitable target can mediate the regression of large, vascularized, metastatic cancers in humans and provide guiding principles as well as encouragement for the further development of immunotherapy for the treatment of patients with cancer.

8) Setting the Body’s ‘Serial Killers’ Loose on Cancer By ANDREW POLLACK AUG. 1, 2016 New York Times Dr. Steven Rosenberg, who turns 76 on Tuesday, still works nearly every day. “I want to end this holocaust,” he said of cancer. Chimeric antigen receptor (CAR) T-cell therapy

Antibody-modified T cells: CARs

10)  Maus, Marcela V., et al. “Antibody-modified T cells: CARs take the front seat for hematologic malignancies.” Blood 123.17 (2014): 2625-2635.
There are 14 publications reporting clinical trials of CAR T cells in hematologic malignancies. All but one of these focused on B-cell malignancies by targeting CD19 or CD20; the other focused on acute myeloid leukemia (AML) by targeting Lewis-Y antigen.

11)  Dai, Hanren, et al. “Chimeric antigen receptors modified T-cells for cancer therapy.” Journal of the National Cancer Institute 108.7 (2016): djv439.  The genetic modification and characterization of T-cells with chimeric antigen receptors (CARs) allow functionally distinct T-cell subsets to recognize specific tumor cells. The incorporation of costimulatory molecules or cytokines can enable engineered T-cells to eliminate tumor cells. CARs are generated by fusing the antigen-binding region of a monoclonal antibody (mAb) or other ligand to membrane-spanning and intracellular-signaling domains. They have recently shown clinical benefit in patients treated with CD19-directed autologous T-cells. Recent successes suggest that the modification of T-cells with CARs could be a powerful approach for developing safe and effective cancer therapeutics.
Adoptive immunotherapy for cancer has a long and somewhat checkered history; the first observations that immune system engagement had antitumor effects are commonly attributed to William Coley, who observed the regression of sarcoma following severe bacterial infections in the 1890s (1). However, the seminal finding that hematopoietic stem cell transplantation (HSCT) using syngeneic donors (from identical twin) was less effective at preventing relapse of leukemia compared with sibling donors provided the founding rationale for adoptive T-cell therapy (2). Additionally, the direct isolation and ex vivo activation of the tumor-infiltrating lymphocytes (TILs) was tested in multiple early-phase studies and resulted in durable responses in melanoma (3).
Recently, laboratory studies of chimeric antigen receptor (CAR)–specific T-cells have been viewed with exceptional interest for clinical development at an array of academic institutions. The redirection of T-cells to tumor antigens by expressing transgenic chimeric antigen receptors takes advantage of potent cellular effector mechanisms via human leukocyte antigen (HLA)–independent recognition. The potential of this approach has recently been demonstrated in clinical trials, wherein T-cells expressing CAR were infused into adult and pediatric patients with B-cell malignancies, neuroblastoma, and sarcoma (4–12).

12) free pdf
Almåsbak, Hilde, Tanja Aarvak, and Mohan C. Vemuri. “CAR T Cell Therapy: A Game Changer in Cancer Treatment.” Journal of Immunology Research 2016 (2016). Almåsbak Hilde CAR T Cell Therapy Game Changer Cancer Immunology Research 2016
The development of novel targeted therapies with acceptable safety profiles is critical to successful cancer outcomes with better survival rates. Immunotherapy offers promising opportunities with the potential to induce sustained remissions in patients with refractory disease. Recent dramatic clinical responses in trials with gene modified T cells expressing chimeric antigen receptors (CARs) in B-cell malignancies have generated great enthusiasm. This therapy might pave the way for a potential paradigm shift
in the way we treat refractory or relapsed cancers. CARs are genetically engineered receptors that combine the specific binding domains from a tumor targeting antibody with T cell signaling domains to allow specifically targeted antibody redirected T cell activation. Despite current successes in hematological cancers, we are only in the beginning of exploring the powerful potential of CAR redirected T cells in the control and elimination of resistant, metastatic, or recurrent nonhematological cancers. This review
discusses the application of the CAR T cell therapy, its challenges, and  strategies for successful clinical and commercial translation.

Years of successive and significant innovations have finally culminated in clinical studies demonstrating the tremendous potential of second generation CAR expressing T cells (Figure 1). Genetic redirection of patient T cells with
CARs targeting the B lymphocyte antigen CD19 has met with exceptional success in various therapy-refractory hematologic diseases (reviewed in [9]). Given their remarkable activity, CAR T cells are expected to enter the mainstream of health care for refractory or relapsed B-cell malignancies within few years and become the game changer for similar approaches in treating other cancers, such as solid tumors.

13)  Journal of Clinical Oncology, 2016 ASCO Annual Meeting (June 3-7, 2016).  Vol 34, No 18_suppl (June 20 Supplement), 2016: LBA3010.  American Society of Clinical Oncology
Anti-CD19 chimeric antigen receptor T cells preceded by low-dose chemotherapy to induce remissions of advanced lymphoma.
James Kochenderfer, Robert Somerville, Tangying Lu, Victoria Shi, James C. Yang, Richard Sherry, Christopher Klebanoff, Udai S. Kammula, Stephanie L. Goff, Adrian Bot, John Rossi, Marika Sherman, Arianne Perez, Allen Xue, Tatyana A. Feldman, Jonathan W. Friedberg, Mark J. Roschewski, Steven Feldman, Lori McIntyre and Steven A. Rosenberg

Background: T cells genetically-modified to express chimeric antigen receptors (CARs) targeting CD19 have potent activity against a variety of B-cell malignancies. Chemotherapy is administered prior to CAR T cells because depletion of recipient leukocytes enhances the anti-malignancy efficacy of adoptively-transferred T cells; an increase in serum interleukin (IL)-15 is one mechanism for this enhancement. Previously, we (Kochenderfer et al. Journal of Clinical Oncology, 2015) and others have reported patients treated with high-dose chemotherapy prior to anti-CD19 CAR T-cell infusions. This report describes treatment of 22 patients with low-dose conditioning chemotherapy followed by infusion of anti-CD19 CAR T-cells. Methods: Eighteen of 22 treated patients received 300 mg/m2 of cyclophosphamide (cy) daily for 3 days; 4 patients received 500 mg/m2 of cy on the same schedule. All patients received fludarabine 30 mg/m2daily for 3 days on the same days as cy. Patients received a single dose of CAR T cells 2 days after completion of chemotherapy. Blood CAR T cells and serum cytokines were analyzed in all patients. Results: Nineteen patients with various subtypes of diffuse large B-cell lymphoma (DLBCL) had the following responses: 8 CR, 5 PR, 2 SD, and 4 PD. One patient with mantle cell lymphoma obtained a CR. Two patients with follicular lymphoma both obtained CRs. Durations of response currently range from 1 to 20 months; 10 remissions are ongoing. All but 4 patients had either chemotherapy-refractory lymphoma or lymphoma that had relapsed after autologous stem cell transplant. The most prominent toxicities were various neurological toxicities. Other toxicities included fever and hypotension. The median peak blood CAR+ cell level was 47/μL (range 4-1217/μL). Patients obtaining CRs or PRs had higher peak blood CAR+ cell levels than patients experiencing SD or PD. The mean serum IL-15 level was 4 pg/mL before the conditioning chemotherapy and 32 pg/mL after chemotherapy (P < 0.0001). Conclusions: Anti-CD19 CAR T cells can induce remissions of advanced B-cell lymphoma when administered after low-dose chemotherapy. In the near future, CAR T cells will likely be a standard therapy for lymphoma.

14)  Brudno, Jennifer N., et al. “Allogeneic T cells that express an anti-CD19 chimeric antigen receptor induce remissions of B-cell malignancies that progress after allogeneic hematopoietic stem-cell transplantation without causing graft-versus-host disease.“ Journal of Clinical Oncology 34.10 (2016): 1112-1121.
We envision a promising future when CAR T-cell therapy will be commonly used in transplant regimens to specifically target malignancy-associated antigens. CAR T cells could be administered as planned infusions along with or soon after stem-cell infusions. Genetically targeted T cells will be an integral part of allogeneic transplant protocols to separate GVM from GVHD.

15)  Heiblig, Maël, Gilles Salles, and Xavier Thomas. “Allogeneic anti-CD19 CAR T cells: new perspectives in the treatment of B-cell malignancies that progress after allogeneic stem cell transplantation?.” Translational Cancer Research 5.1 (2016): S5-S8.

16) Heijink, D. M., et al. “T-cells fighting B-cell lympho proliferative malignancies: the emerging field of CD19 CAR T-cell therapy.”Heijink B_Cell lymphoproliferative malignancies CD19 CAR T_Cell 2016
CD19 CAR T-cells specifically target CD19 positive B-cells, including healthy
B-cells, resulting in long lasting B-cell aplasia. This aplasia – a surrogate for the persistence of CAR T-cells – puts successfully treated patients at risk for infections.

17) CAR T-Cell Therapy The Road Ahead Rachel Webster August 11, 2016
BioProcess Online

Remarkable early-phase clinical data for CAR T-cell therapy was first announced in 2014, and since then excitement has been growing. CAR T-cell therapies have achieved impressive complete response (CR) rates of up to 90% in pediatric and adult patients with relapsed or refractory (R/R) acute lymphocytic leukemia (ALL) and non-Hodgkin’s lymphoma (NHL); durable remissions and six-month survival of more than 70% have been reported.
Off-target sides effects associated with CAR T-cell therapy, namely cytokine release syndrome (CRS), are a major concern. Severe (and potentially fatal) CRS has been observed in approximately one-third of R/R ALL patients. A high-risk of CRS could confine CAR T-cell approaches to late-line treatment settings, when other treatments have been exhausted.
The first CAR T-cell therapy could enter the market in 2017.

18) Rock star cancer treatment is being scrutinized after clinical trial deaths
Jamie Reno, Yahoo Finance Contributor July 31, 2016
In 2014, Juno’s CAR-T clinical trials were stopped temporarily at Memorial Sloan Kettering Cancer Center in New York due to several patient deaths as a result of cytokine-release syndrome (CRS), a potentially deadly toxicity associated with increased levels of cytokines in the body including interleukin (IL)-6 and interferon γ.

19) UPDATED: Racing past a wounded Juno, Kite aims to file lead CAR-T for OK by end of 2016  by John Carroll August 8, 2016
Less than two months ago, Kite held a ribbon cutting ceremony for its new manufacturing facility, a 43,500-square-foot plant that will be used to make its personalized KTE-C19.
“Our commercial facility will have the capacity to produce up to 5,000 patient therapies per year and we expect it to be operational in producing clinical materials by year-end,” noted Belldegrun. “Overall, we have continuously been optimizing key aspect of our manufacturing, supply chain, and quality control and possess a proprietary process that dramatically reduces the time to approximately 14 days for when a patients material are shift to our facility to when the engineered T-cells are released to the patient. This is one of the fastest rates in the industry.”

20)   Kite looks to hammer home advantage over Juno with quick CAR-T approval, launch  by Nick Paul Taylor | Aug 9, 2016

If Kite achieves this goal, it will position itself to win approval for KTE-C19 as a treatment for diffuse large B-cell lymphoma, primary mediastinal B cell lymphoma and transformed follicular lymphoma next year. With the brief clinical hold causing Juno’s targeted approval to slip from 2017 to 2018, Kite is positioned to come to market well before its rival, while also pipping Novartis ($NVS) to the post.
Asked how soon after approval Kite would be ready to launch KTE-C19, Chief Commercial Officer Shawn Tomasello was unequivocal.
“Immediately. We’ll be ready,” Tomasello said.
To live up to Tomasello’s bullishness, Kite will need to have all its commercial and logistic units ready to go by some time next year. Work in these areas is already well underway. Tomasello is in the middle of building out a commercial team that will target 50 to 70 cancer centers.

21)  T-cell therapies for cancer: from outsider to pharmaceutical darling
The Pharmaceutical Journal 24 AUG 2016 By Janna Lawrence
there are now three competing companies. And all of them are working on cell therapies that target CD19 on B cells.
The treatment of B cell cancers has been so successful because they are the only cells that express CD19 and also because a person can live without B cells.

22)  Davila, Marco L., and Michel Sadelain. “Biology and clinical application of CAR T cells for B cell malignancies.” International journal of hematology (2016): 1-12.

23) full pdf
Miller, Brian C., and Marcela V. Maus. “CD19-Targeted CAR T Cells: A New Tool in the Fight against B Cell Malignancies.” Oncology research and treatment 38.12 (2015): 683-690. CD19 CAR T Cells Against B Cell Malignancies Oncology research Miller Brian 2015

24) May, Megan Brafford, and Melissa Olsommer. “Emerging Treatment Options Utilizing Chimeric Antigen Receptor T-Cells.” Journal of Clinical & Cellular Immunology 2016.

25) full pdf
Heijink, D. M., et al. “T-cells fighting B-cell lympho proliferative malignancies: the emerging field of CD19 CAR T-cell therapy.” Netherlands Journal of Medicine May (2016): 147. T cells fighting B-cell malignancies CD19 CAR T-cell therapy Heijink The Netherlands Journal of Medicine MAY 2016

26)  Immunotherapy of non-Hodgkin’s lymphoma with a defined ratio of CD8+ and CD4+ CD19-specific chimeric antigen receptor–modified T cells
Science Translational Medicine 07 Sep 2016: Vol. 8, Issue 355, pp. 355ra116      Cameron J. Turtle1,2,*, Laïla-Aïcha Hanafi1, Carolina Berger1,2, Michael Hudecek1,†, Barbara Pender1, Emily Robinson1, Reed Hawkins1, Colette Chaney1, Sindhu Cherian3, Xueyan Chen3, Lorinda Soma3, Brent Wood3, Daniel Li4, Shelly Heimfeld1, Stanley R. Riddell1,2,‡ and David G. Maloney1,2,‡  1Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

27)  Onea, Alexandra S., and Ali R. Jazirehi. “CD19 chimeric antigen receptor (CD19 CAR)-redirected adoptive T-cell immunotherapy for the treatment of relapsed or refractory B-cell Non-Hodgkin’s Lymphomas.” American journal of cancer research 6.2 (2016): 403.

Addition of Ibrutinib to CAR T-Cells Improves Outcome in Mantle Cell Lymphoma,  Stephen J. Schuster,  U Penn

28) 704 The Addition of the BTK Inhibitor Ibrutinib to Anti-CD19 Chimeric Antigen Receptor T Cells (CART19) Improves Engraftment and Antitumor Responses Against Mantle Cell Lymphoma
Lymphoma: Pre-Clinical – Chemotherapy and Biologic Agents
Program: Oral and Poster Abstracts  Type: Oral
Session: 625. Lymphoma: Pre-Clinical – Chemotherapy and Biologic Agents: B-cell Receptor Signaling and Resistance in Lymphoma
Monday, December 7, 2015: 3:00 PM  Hall E2, Level 2 (Orange County Convention Center)

Marco Ruella, MD1, Saad S Kenderian, MD1, Olga Shestova, PhD1*, Joseph A. Fraietta, PhD1*, Sohail Qayyum, MD2*, Qian Zhang, MD, PhD2*, Marcela V. Maus, MD, PhD1, Xiaobin Liu, PhD2*, Selene Nunez-Cruz, PhD1*, Michael Klichinsky, PharmD1*, Omkar Uday Kawalekar, MS, BS1, Michael C. Milone, MD, PhD1*, Simon F. Lacey, PhD, BSc1*, Anthony Mato, MD3*, Stephen J. Schuster, MD4, Michael Kalos, PhD2, Carl H. June, MD1, Saar I Gill, MBBS, PhD1 and Mariusz A. Wasik, MD5*

1Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, PA 2Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 3Center for Chronic Lymphocytic Leukemia, Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA 4Division of Hematology-Oncology, University of Pennsylvania, Philadelphia, PA
5Pathology and Laboratory Medicine, Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA
Introduction: The bruton tyrosine kinase (BTK) inhibitor ibrutinib demonstrates considerable activity in mantle cell lymphoma (MCL). However, approximately 30% of patients do not respond to this treatment and the therapy invariably leads to drug resistance with a median response of 17.5 months. Infusion of autologous T cells transduced with chimeric antigen receptors (CAR) against the B-cell specific CD19 antigen (CART19) leads to dramatic clinical responses in the majority of patients with acute lymphoblastic leukemia and the activity in B cell lymphoma is currently being evaluated in clinical trials. Bulky disease, as sometimes seen in MCL, may impair T cell infiltration. The features of ibrutinib that make it an interesting addition to CART19 include its efficacy in reducing tumor masses and its ability to mobilize neoplastic B cells into the peripheral blood, thereby potentially exposing them to the killing activity of CART19. Therefore, we sought to investigate the combination of the two novel targeted therapies, ibrutinib and CART19 in MCL.

Results: In vitro studies with established MCL cell lines and with a novel cell line (MCL-RL) showed a range of responses to ibrutinib with an IC50 ranging from 10 nM to 10 µM; MCL-RL was the most sensitive cell line evaluated with an IC50 of 10nM, similar to primary MCL. Both ibrutinib-sensitive and ibrutinib-resistant cell lines strongly activated CART19 in an antigen-specific manner as detected by CD107a degranulation, cytokine production and CFSE proliferation assays. Importantly, in vitro assays with MCL cell lines co-cultured with increasing doses of CART19 (E:T= 2:1, 1:1, 0.5:1, 0.25:1) combined with increasing concentrations of ibrutinib (0, 10, 100, 1000 nM) demonstrated strong additive tumor killing (Figure 1). Notably, supra-therapeutic doses of Ibrutinib (>/=1 uM) impaired cytokine production and T cell proliferation in vitro. In order to test this combination in vivo we established a novel MCL model, injecting i.v. luciferase-positive MCL-RL cells into NSG mice. This resulted in 100% MCL engraftment in liver and spleen, with eventual dissemination into lymph nodes and bone marrow. Treatment with three different doses of CART19 (0.5, 1 and 2 million cells/mouse) led to a dose dependent anti-tumor effect. A similar dose response to CART19 was also observed in the ibrutinib-resistant Jeko-1 cell line. We also treated MCL-RL xenografts with different doses (0, 25 and 125 mg/Kg/day) of ibrutinib, with a median overall survival respectively of 70, 81 and 100 days (p<0.001). Importantly, a direct in vivo comparison of the highest ibrutinib dose (125 mg/kg) and CART19 showed a significantly improved tumor control for mice treated with CART19. However, treatment with either CART19 or ibrutinib as single agents invariably led to late relapse. Therefore we sought to treat MCL-RL xenografts with the combination of CART19 and ibrutinib and compare it to the single agent activity. The combination resulted in significant improvement in tumor control compared to mice treated with the single agents with 80% of mice achieving long-term disease-free survival (p=0.007 at day 110, representative mice shown in Figure 2A). Intriguingly, we found that mice treated with ibrutinib had higher numbers of circulating CART19 cells (Figure 2B).

Conclusions: Combining CART19 with ibrutinib represents a rational way to incorporate two of the most recent therapies in MCL. Our findings pave the way to a two-pronged therapeutic strategy in patients with MCL and other types of B-cell lymphoma.

28) Ramos CA, Dotti G. Chimeric Antigen Receptor (CAR)-Engineered Lymphocytes for Cancer Therapy. Expert opinion on biological therapy. 2011;11(7):855-873.

29) J Immunol. 2015 Aug 1;195(3):755-61. Chimeric Antigen Receptor- and TCR-Modified T Cells Enter Main Street and Wall Street. Barrett DM1, Grupp SA2, June CH3.

The field of adoptive cell transfer (ACT) is currently comprised of chimeric Ag receptor (CAR)- and TCR-engineered T cells and has emerged from principles of basic immunology to paradigm-shifting clinical immunotherapy. ACT of T cells engineered to express artificial receptors that target cells of choice is an exciting new approach for cancer, and it holds equal promise for chronic infection and autoimmunity. Using principles of synthetic biology, advances in immunology, and genetic engineering have made it possible to generate human T cells that display desired specificities and enhanced functionalities. Clinical trials in patients with advanced B cell leukemias and lymphomas treated with CD19-specific CAR T cells have induced durable remissions in adults and children. The prospects for the widespread availability of engineered T cells have changed dramatically given the recent entry of the pharmaceutical industry to this arena. In this overview, we discuss some of the challenges and opportunities that face the field of ACT.

30) Power Point Slide Presentation:
Chimeric_Antigen_Receptor_T-cell_Power_Point

31) Kochenderfer, James N., et al. “Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor.” Journal of Clinical Oncology 33.6 (2015): 540-549.  Lymphoma Can Be Effectively Treated With Anti-CD19 Chimeric Antigen ReceptorJames Kochenderfer

Ibrutinib effective for CNS Relapse in Mantle Cell and B-Cell Lymphoma

Ibrutinib Crosses BBB

32) Bernard, Sophie, et al. “Activity of ibrutinib in mantle cell lymphoma patients with central nervous system relapse.” Blood 126.14 (2015): 1695-1698.

33) Falchi, Lorenzo, et al. “BCR Signaling Inhibitors: an Overview of Toxicities Associated with Ibrutinib and Idelalisib in Patients with Chronic Lymphocytic Leukemia.” Mediterranean journal of hematology and infectious diseases 8.1 (2016).

34)   Acalabrutinib, a selective second-generation BTK inhibitor Jingjing Wu, Journal of Hematology & Oncology20169:21  Acalabrutinib (ACP-196) is a novel irreversible second-generation BTK inhibitor that was shown to be more potent and selective than ibrutinib.

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