OOTR the 8th Annual Conference Satellite Seminar (Co-organised by Eisai Co., Ltd.)
Eric P. Winer / Ian E. Krop (Harvard University) Seminar
"Late recurrence of ER+breast cancer: Unresolved clinical challenge" / "Novel approaches to HER2-positive metastatic breast cancer"
(19th April, 2012 Tokyo Station Conference, Tokyo)

Dr. Krop
Dr. Krop

At the satellite seminar of OOTR the 8th Annual Conference there were a total of 70 participants which greatly exceeded the expected attendance of 40.

Dr. Ian Krop, using mainly clinical data regarding Anti-HER2 therapy drugs such as Pertuzumab, T-DM1 and Neratinib, which is currently in the process of development, lectured on the comprehensive current status as well as future prospects.
He also presented a flow of near future first line to third line within this subset.

Dr. Eric Winer lectured on late recurrence hormone positive breast cancer, which occurs after 5 years and later, which is currently one of the most important topics. He explained about the issues and modalities of responses and tests in the future.

Dr. Winer
Dr. Winer

There were many questions from the participants and both doctors who lectured answered the questions passionately, sometimes with humour, which left a lasting impression.

Thank you very much to both doctors who gave us their lectures, Dr. Jinno of Keio University, who acted as the moderator for both sessions, and to all the doctors and medical professionals who participated, some from as far away as Nagoya.(Reported by Dr. Shigehira Saji, Kyoto University)

Kyoto Academic Lecture Presentation Sponsored by OOTR
'From Scientific Research to Clinical Application of Cancer Antibody Therapy'

Nagoya City University School of Medical Sciences
specially appointed Professor Ryuzo Ueda
(November 9th 2010 at Hotel Granvia Kyoto)

From Scientific Research to Clinical Application of Cancer Antibody Therapy

OOTR sponsored the 'Kyoto Academic Lecture Presentation Meeting' of the 'Cancer Professional Training Plan' which was held on November 9th 2010. We would like to introduce the details of the special lecture given by Professor Ryuzo Ueda of Nagoya City University School of Medical Sciences who is the specially appointed professor: 'From Scientific Research to Clinical Application of Cancer Antibody Therapy':

Adult T-cell leukemia/lymphoma (ATLL), HTLV- I-associated myelopathy (HAM)

This year, the decision was made that antibody testing of Human T-cell leukemia virus Type 1(HTLV-1), which is the causal virus for Adult T-cell leukemia/lymphoma (ATLL) and HTLV- I-associated myelopathy (HAM), using public funds, is to be added to prenatal checkups. ATLL (called ATL previously) is a disease identified in 1977, and in 1980 it became clear that it is an infection caused by a retrovirus. Transovarial transmission from mother to child was also proved and the frequency of such transmission has gone down by restricting lactocation from mothers with HTLV-1 antibody positive (the carrier) to their child. It is estimated that the number of HTLV-1 infected carriers is currently around 1,100,000 persons and 3-5% of them develop ATL and 0.3% develop HAM (Table 3). The major treatment is chemotherapy but many cases are non responsive. There are reports that mini-transplant is effective for young ATLL patients but in many cases they develop the disease at an advanced age and there is no established treatment method.

In adult T-cell leukemia/lymphoma (ATLL), the expression of CCR4, which is a chimokine receptor, is strongly positive for more than 90% of tumors. We have developed a novel antibody and found effectiveness in clinical trials and researches as shown in Table 4.

Table 3
Table 3
Table 4
Table 4

1. ATLL and CCR4

CCR4 is a 7 transmembrane G protein-coupled receptor and exists at chromosome 3p. For ligands, there are CCL17 (TARC), CCL22 (MDC) (Table 5). At normative T cells, there are various functions but also within ATLL there exist tumors which retain the function of normal regulatory T cells (Treg) (Table 6). The CCR4 antibody hardly shows cross reactivity with normal organs and it only reacts with T helper 2 cells (Th2) of normal lymphocyte and fractionation of regulatory T cells.

Table 5
Table 5
Table 6
Table 6

2. Clinical condition of ATL from the view point of CCR4

In breast cancer, many metastases can be found in lungs and bone but metastasis to kidneys is rare. It seems that the interaction between CXCR4 is important, because the receptor and the ligand are closely associated with metastasis. In the ATLL cases with derma infiltrates, all the tumors are CCR4 positive. CCR4 are expressed at Th2 and Treg cell fractionation, however the phenotypes of normal Treg are CD4 and CD25 positive and they are common with ATLL cells. There is FoxP3, a transcription factor as a specific molecular marker for Treg cells but as the result of expression analysis, both at mRNA level and at protein level, are strongly expressed, suggesting that ATLL's outset metrocyte is Treg cell. In ATLL cases where immunocompromised status is developed, there are approximately 1/3 of cases where Treg cell's function is retained. It seems that they are working suppressively against cellular proliferative potential and IFNγ productivity (Table 9).

Table 9
Table 9

3. Clinical Importance of ATLL which expresses CCR4

Whether an ATLL or a Peripheral T-Cell Lymphoma/Unspecified (PTCL-u), the existence period of CCR4 positive cases are a lot shorter when compared with CCR4 negative cases. Analysis of Aichi Cancer Center Laboratory's PTCL-u cases showed that groups of unstable genes have poor prognosis and the expression of CCR4 is highly frequent.

4. CCR4 antibody as ATLL curative medicine

The action mechanism of antibody therapy against malignant tumors can be pointed out from; 1) that it joins the tumor cell and implements apoptosis induction 2) it acts with alexin complements and destroys tumor cells 3) through antibodies it induces NK-cells and macrophage and destroys carcinolysis (ADCC). From the viewpoint of clinical effectiveness, ADCC activity is especially important for the induction of anticancer efficacy. ADCC activity is, in the antibodies where fucose of sugar chain at the Fc part of the antibody is removed, compared with normal antibodies made from CHO cells, it indicated the ADCC activity goes from 100 times to 1000 times effectiveness. Dr. Matsushima (currently The University of Tokyo, preventive medicine) et al, by utilizing the result of CCR4 gene cloning, they have succeeded in creating an antibody where the distal N of CCR4 as epitope at former Kyowa Hakko Kogyo Tokyo Laboratory. Then they created defucosylated antibodies and chimera antibodies for clinical use and also created Human type CCR4 antibodies and started the clinical tests. Having confirmed ADCC activity from patients' specimens and proved ADCC activity of NK-cells against tumor cells in vitro (The world's first data).

The defucosylated antibodies eminently enhanced ADCC activity. When the ATL cell line (S-Yu) cells which were successfully established at Kyoto University in Professor Uchiyama's Department (currently Professor Takaori's Department) was transplanted to an SCID mouse, the effect of tumor shrinkage was observed not just with antibodies, but especially when administered together with G-CSF then the tumor totally vanished and lead to a cure. It was proved that this is reproducible at CCR4 positive PTCL-u and that the antibodies can be used as a curable medicine.

5. Creating an immunotherapy model

When implanting human femur under the skin of an SCID mouse (SCID-hu mouse) and administering human bone-marrow cells into it, the hematopoietic cells of humans propagate/differentiate. This can be subcultured to other SCID-hu mice but there is the ethical issue of using the bone of fetuses therefore we have created successfully the ATLL model by using the NOD/Shi-scid, IL-2Rγnull mouse (NOG mouse) which was developed in Japan recently. NOG mouse is a severe combined immunodeficiency mouse and in addition to T/B cells, the NK-cells and macrophage function are also imminently descended and it is capable of the engraftment of tumor cells taken from patients' or differentiation and propagation of human cells. By implanting the human immune charge cells to an NOG mouse, the reproduction of the immune environment similar to that of humans can be reproduced. Similar clinical conditions were recreated to those of the NOG mouse from the tumor cells of Hodgkin's disease, acute lymphoblastic lymphoma (LBL) and ATLL patients. When the tumors were treated with CCR4 antibodies, the ATLL cells disappeared and proved that the NK cells of the patients were able to be eradicated with the ADCC active antibodies with the patients' own ATLL cell antibody dosage.

6. Domestically grown antibody treatment against cancer for the first time ever in Japan

Based on the results of previous clinical research, we have started to verify that the CCR4 antibody is effective. Not chimera type antibodies, but we left the amino-acid sequence which identify only the antigen determinant with mouse antibody and the rest of antibody elements can be recreated at the same previous clinical test results whether it is the whole human type of humanized antibody. We have completed verification of its stability as a medicine using rhesus macaques. We decided to use it as a medicine for treatment purposes.

6-1. Decision of medical candidates and treatment schedule

For the phase 1 trial on humans, we chose medical candidates who were patients of ATLL or PTCL-u of CCR4 positive and those with medical cases that have relapsed after chemotherapy. An intravenous dosage of 4 times per week was given and the judgment of effectiveness was within 4 weeks after the last dosage. Deciding on the first dosage amount was difficult, but we made the first dosage amount to be 0.01mg/kg. This amount was agreed on by taking into consideration the fact that even with the low density of 1/100 defucosylated antibody it still retains the same degree of ADCC active of the normal antibody and the effectiveness won't be expected unless the amount is to be effective against the normal Th2 cells and Treg cells of CCR4 positive. Observing the existence or non existence of side-effects, we increased the amount by degrees up to 1mg/kg. We didn't recognize the cases where heavy side-effects or strong toxicity occurred. We made the Phase 2 trial's recommended amount to be 1.0mg/kg. There existed no antibody against the administered antibody and it was suggested that multiple administering is possible.

6-2. The result of the Phase 1 trial

Case 1. 68 year old, female. Chemotherapy was implemented but she relapsed. ATLL cells of about 20% within the peripheral blood was detected. By administering the CCR4 antibody at 0.01mg/kg, the ATLL cells disappeared within the peripheral blood the following morning. Several percentage was detected after one week however but it disappeared again the following day after the treatment and it was judged a complete remission.

Case 2. 60 year old, male. Chemotherapy was intensively implemented but recognized the dermal infiltration and punched-out lesion. With a dosage of 4 times and after 3 weeks it was judged that the dermal infiltration and punched-out lesion had improved and no change (SD). After 1 year, the dermal infiltration and punched-out lesion had disappeared and there was complete remission. For about 3 years there has been no relapse.

Case 3. 64 year old, female. PTCL cell appeared in the peripheral blood and there was an enlarged inguinal lymph node. After the first treatment, PTCL cells within the peripheral blood and exanthema had both disappeared and the inguinal lymph node was impalpable (remained on CT). It was judged a partial remission (PR) and the judgment on the whole is PD.

Of the 16 registered cases, the responsive cases at the time of the judgment were 5 cases (31.3%) and complete remission cases were 2 cases and no exacerbation after that has been recognized (Table 26 and 27).

The Phase 2 trial started from May 2009 to the ATLL of chemotherapy nonresponsive with 1mg/kg of the dosage of 8 times and the registration has already completed. Out of the 26 cases which are capable of evaluation, 13 cases (50%) showed the effectiveness and the result has been adopted at a lecture at The American Society of Hematology (ASH) which will be held in December this year.

6-3. The importance of Reverse Translational Research (R-TR)

The important research tasks are to research the various causes of the effective cases and non-effective cases. In the eminent cases research the reason why the treatment effect can last with only a small amount of antibody. In exanthema cases, research its cause and the difference of required time till the effectiveness occurs and research the action mechanism. The research of the influence the CCR4 antibody gives to the normal Treg cells is also a task and that the Reverse-TR is important for drawing up a future optimum therapy plan. We are in preparation right now, aiming for the application of approval from the Pharmaceuticals and Medical Devices Agency (PMDA) as an orphan drug in the spring of 2011.

Table 26
Table 26
Table 27
Table 27

7. The meaning of CCR4 on tumor immunity

Since ATLL cells resemble normal Treg cells and expression type, and some share the same functions, it is suggested that HTLV-1 virus persistently infects the Treg cells of CCR4 positive and influences the occurrence of ATL.

At B-cell Hodgkin's lymphoma, it became clear that TARC and MDC are abundant in the culture supernatant of the cell lines. When double dyed the antibodies of the cells around Hodgkin's cell with CCR4 antibody and FoxP3 antibody, it was observed there were many lymphocyte of double positives. Treg cells work suppressively against CTL which targets Hodgkin's cells. If the environment can be prepared where CTL is easily active by removing Treg cells by administrating CCR4 antibodies, it enables the development of a new treatment. Especially it is considered to be effective in the region of antigen-specific immune therapy.

8. Control of Treg cells on tumor immune therapy

In order to make immune therapy effective in clinical practice also, it will be important to improve cancer vaccination and how to devise an administration vaccine to reach the focus. At the focus a device is necessary in order to increase CTL cells, which are specific to cancer and also highly active. However around the tumor exist Treg cells too therefore if they are heavily induced, CTL's function will be restrained and the effect of vaccine cannot be anticipated sufficiently. On this point, the removal of Treg cells from the microenvironment of the tumor with the before mentioned CCR4 antibody etc will be an anticipated method. That FDA in the USA approved the tumor lysate-pulsed dendritic cell (Sipuleucel-T) for prostate cancer in April this year can be said that the first year of abscess immune therapy has arrived.


In TR, it's not just the clearing up of mechanism but it is important that the patient gets an outcome returned that is a benefit. It should be addressed that TR will evolve and lead to therapy development only when harmonious joint research is implemented in conjunction with quality foundation research, clinical research with deep insight and highly willing cooperation.

Lecture of Prof. Aaron Ciechanover (Laureate of Nobel Prize in chemistry)
"Why our proteins have to die so we shall live?" (9 November, 2007)

Prof. Aaron Ciechanover
Prof. Aaron Ciechanover

On the November 9th, 2007, Dr. Aaron Ciechanover, the professor of Vascular and Cancer Biology Research Center, The Rappaport Faculty of Medicine and Reserch Institute and Technion-Israel Institute of Technology, delivered a lecture entitled 'Why our proteins have to die so we shall live?' Many undergraduate and graduate students and medical doctors attended this special lecture. In the beginning, the professor Kohei Shiota, the dean of graduate school of medicine in Kyoto University, introduced professor Ciechanover. Dr. Masakazu Toi, the professor of the department of breast surgery, acted as a master of the lecture.

Professor Ciechanover started with the history of protein research. For a long while, it had been considered that proteins in our body wouldn't be changed but static. Nevertheless, the conceptual changes occurred gradually since Dr. Rudolf Schoenheimer, who is the father of protein research field, discovered a method to label proteins with heavy isotope-labeled amino acids. Fifteen years later, Dr. David Hogness and Dr. Jacques Monod found that proteins within mammalian cells are dynamic. In 1955, Dr. Christian de Duve discovered the lysosome, which brought a significant change of the concept, because the cellular proteins were shown to be degraded in the lysosomes. Then, in mid-70's, Dr. Brian Poole showed that intracellular proteins were also degraded in non-lysosomal machinery, which resulted in the discovery of ubiquitin system. In 1976, Dr. Ciechanover began intensive researches on the non-lysosomal proteolytic system.

Dr. Ciechanover's group found that three enzymes E1, E2, and E3 are involved in the proteolysis. Ubiquitin is activated by the ubiquitin-activating enzyme (E1). An ubiquitin-conjugating enzyme (E2) transfers the activated ubiquitin moiety to the protein substrate that is bound specifically to a unique ubiquitin ligase (E3). Successive conjugation of ubiquitin moieties to one another generates a polyubiquitin chain. Unless protein substrates are marked by a polyubiquitin chain, proteases are unable to recognize.

The audience was listening intently
The audience was listening intently

It is known that the ubiquitin system provides many functions, such as control of cell cycle-associated factors, regulation of signaling factors or transcriptional factors. Thereby, ubiquitin is directly involved in cell proliferation, cell death and cell differentiation. Accumulation of proteins conjugated to ubiquitin causes a variety of neurodegenerative disorders, such as Alzheimer disease, Parkinson disease, Angelman syndrome and Prader-Willi syndrome. Moreover, ubiquitin system closely links with cancer development, proliferation, invasion and resistance. In fact, ubiquitin protease inhibitor has been developed for anticancer drugs. For Instance, Bortezomib, which is able to regulate the accumulation of the abnormal proteins and inactivation of NF-kB, improves the treatment of multiple myeloma, refractory follicular non-Hodgekin lymphoma and other malignant diseases.

In the end, professor Ciechanover introduced his background that he had worked as a general surgeon about 30 years ago. Then, he concluded that 'from bench to bedside' is essentially important to clarify the mechanism of diseases, although it requires a great investment of time and effort. He also emphasized that the research outcomes must be returned to society. (Reported by Dr. Wakako Tsuji, Kyoto University)

Aaron Ciechanover Technion-Israel Institute of Technology

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