Society for Immunotherapy of Cancer clinical and biomarkers data sharing resource document: Volume II-practical challenges.

The development of strongly predictive validated biomarkers is essential for the field of immuno-oncology (IO) to advance. The highly complex, multifactorial data sets required to develop these biomarkers necessitate effective, responsible data-sharing efforts in order to maximize the scientific knowledge and utility gained from their collection. While the sharing of clinical- and safety-related trial data has already been streamlined to a large extent, the sharing of biomarker-aimed clinical trial derived data and data sets has been met with a number of hurdles that have impaired the progression of biomarkers from hypothesis to clinical use. These hurdles include technical challenges associated with the infrastructure, technology, workforce, and sustainability required for clinical biomarker data sharing. To provide guidance and assist in the navigation of these challenges, the Society for Immunotherapy of Cancer (SITC) Biomarkers Committee convened to outline the challenges that researchers currently face, both at the conceptual level (Volume I) and at the technical level (Volume II). The committee also suggests possible solutions to these problems in the form of professional standards and harmonized requirements for data sharing, assisting in continued progress toward effective, clinically relevant biomarkers in the IO setting.

Society for Immunotherapy of Cancer clinical and biomarkers data sharing resource document: Volume I-conceptual challenges.

The sharing of clinical trial data and biomarker data sets among the scientific community, whether the data originates from pharmaceutical companies or academic institutions, is of critical importance to enable the development of new and improved cancer immunotherapy modalities. Through data sharing, a better understanding of current therapies in terms of their efficacy, safety and biomarker data profiles can be achieved. However, the sharing of these data sets involves a number of stakeholder groups including patients, researchers, private industry, scientific journals and professional societies. Each of these stakeholder groups has differing interests in the use and sharing of clinical trial and biomarker data, and the conflicts caused by these differing interests represent significant obstacles to effective, widespread sharing of data. Thus, the Society for Immunotherapy of Cancer (SITC) Biomarkers Committee convened to identify the current barriers to biomarker data sharing in immuno-oncology (IO) and to help in establishing professional standards for the responsible sharing of clinical trial data. The conclusions of the committee are described in two position papers: Volume I-conceptual challenges and Volume II-practical challenges, the first of which is presented in this manuscript. Additionally, the committee suggests actions by key stakeholders in the field (including organizations and professional societies) as the best path forward, encouraging the cultural shift needed to ensure responsible data sharing in the IO research setting.

Tumor-Associated Disialylated Glycosphingolipid Antigen-Revealing Antibodies Found in Melanoma Patients' Immunoglobulin Repertoire Suggest a Two-Direction Regulation Mechanism Between Immune B Cells and the Tumor.

There is far less information available about the tumor infiltrating B (TIL-B) cells, than about the tumor infiltrating T cells. We focused on discovering the features and potential role of B lymphocytes in solid tumors. Our project aimed to develop innovative strategies to define cancer membrane structures. We chose two solid tumor types, with variable to considerable B cell infiltration. The strategy we set up with invasive breast carcinoma, showing medullary features, has been introduced and standardized in metastatic melanoma. After detecting B lymphocytes by immunohistochemistry, VH-JH, Vκ-Jκ immunoglobulin rearranged V region genes were amplified by RT-PCR, from TIL-B cDNA. Immunoglobulin variable-region genes of interest were cloned, sequenced, and subjected to a comparative DNA analysis. Single-chain variable (scFv) antibody construction was performed in selected cases to generate a scFv library and to test tumor binding capacity. DNA sequence analysis revealed an overrepresented VH3-1 cluster, represented both in the breast cancer and the melanoma TIL-B immunoglobulin repertoire. We observed that our previously defined anti GD3 ganglioside-binder antibody-variable region genes were present in melanoma as well. Our antibody fragments showed binding potential to disialylated glycosphingolipids (GD3 ganglioside) and their O acetylated forms on melanoma cancer cells. We conclude that our results have a considerable tumor immunological impact, as they reveal the power of TIL-B cells to recognize strong tumor-associated glycosphingolipid structures on melanomas and other solid tumors. As tumor-derived gangliosides affect immune cell functions and reduce the B lymphocytes' antibody production, we suspect an important B lymphocyte and cancer cell crosstalk mechanism. We not only described the isolation and specificity testing of the tumor infiltrating B cells, but also showed the TIL-B cells' highly tumor-associated GD3 ganglioside-revealing potential in melanomas. The present data help to identify new cancer-associated biomarkers that may serve for novel cancer diagnostics. The two-direction regulation mechanism between immune B cells and the tumor could eventually be developed into an innovative cancer treatment strategy.

Challenging tumour immunological techniques that help to track cancer stem cells in malignant melanomas and other solid tumours.

AIM OF THE STUDY: The arsenal of questions and answers about the minor cancer initiating cancer stem cell (CSC) population put responsible for cancer invasiveness and metastases, has left with an unsolved puzzle. Specific aims of a complex project were partly focused on revealing new biomarkers of cancer. We designed and set up novel techniques to facilitate the detection of cancerous cells. MATERIALS AND METHODS: As a novel approach, we investigated B cells infiltrating breast carcinomas and melanomas (TIL-B) in terms of their tumour antigen binding potential. By developing the TIL-B phage display technology we provide here a new technology for the specific detection of highly tumour-associated antigens. Single chain Fv (scFv) antibody fragment phage ELISA, immunofluorescence (IF) FACS analysis, chamber slide technique with IF confocal laser microscopy and immunohistochemistry (IHC) in paraffin-embedded tissue sections were set up and standardized. RESULTS: We showed strong tumour-associated disialylated glycosphingolipid expression levels on various cancer cells using scFv antibody fragments, generated previously by uniquely invasive breast carcinoma TIL-B phage display library technology. CONCLUSIONS: We report herein a novel strategy to obtain antibody fragments of human origin that recognise tumour-associated ganglioside antigens. Our investigations have the power to detect privileged molecules in cancer progression, invasiveness, and metastases. The technical achievements of this study are being harnessed for early diagnostics and effective cancer therapeutics.

[Genetic information from tumor-infiltrating B lymphocytes as a driver tool ("GPS") for anti-tumor T cell CARs]. / A tumorban felhalmozódó B-limfociták genetikai információja, mint az immun T-sejt vezette tumorellenes "autó" (CAR, kiméra antigénreceptor) célba irányító "GPS"-e.

The rapidly growing field of gene therapy techniques to modify T cells with chimeric antigen receptors (CARs) for cancer care solutions, reached considerable achievements. However, there is an urgent need of reliable, well tolerable tumor-associated antigen specific antibodies. Tumor-infiltrating B (TIL-B) cell originated single chain Fv (scFv) gene regions could be selected with tumor specificity. DNA sequences of these antibody variable regions were subjects to get engineered into new CAR constructs. Our novel strategy harnesses tumor-infiltrating B cells' unique capacity to reveal highly tumor-associated disialylated glycosphingolipids (GD3 gangliosides). We used these human antibody fragments for generating GD3 ganglioside specific CAR gene constructs for potential usage in solid tumors.

The novel panel assay to define tumor-associated antigen-binding antibodies in patients with metastatic melanomas may have diagnostic value.

We aim to harness the natural humoral immune response by various technologies to get novel biomarkers. A complex antibody analysis in sera and in the tumor microenvironment leads to reveal tumor-specific antibodies. More strategies were introduced to select the most effective one to identify potential tumor antigen-binding capacity of the host. Epstein-Barr virus transformation and cloning with limiting dilution assay, magnetic cell sorting and antibody phage display with further methodological improvements were used in epithelial and neuroectodermal cancers. Column-purified sera of patient with melanoma were tested by immunofluorescence assay, while sera of further melanoma patients were processed for membrane-binding enzyme-linked immunosorbent assay. Some supernatants of selected B cell clones and purified antibodies showed considerable cancer cell binding capacity by immunofluorescence FACS analysis and confocal laser microscopy. Our native tumor cell membrane preparations helped to test soluble scFv and patients' sera for tumor binder antibodies. A complex tumor immunological study was introduced for patients with melanoma (ethical permission: ETT TUKEB 16462-02/2010); peripheral blood (n = 57) and surgically removed primary or metastatic tumors (n = 44) were gathered and processed at cellular immunological level. The technological developments proved to be important steps forward to the next antibody profile analyses at DNA sequence level. Cancer cell binding of patient-derived antibodies and natural immunoglobulin preparations of pooled plasma product intravenous immunoglobulins support the importance of natural human antibodies. Important cancer diagnostics and novel anticancer strategies are going to be built on these tools.

Conference overview: Cancer Immunotherapy and Immunomonitoring (CITIM): moving forward.

The immune system is a critical element involved in the control of tumor development and progression. While professionals have learned how to manipulate the immune system to generate tumor-specific immune responses, cancer immunotherapy has not yet delivered substantial clinical benefits. It has become increasingly clear that tumor-induced abnormalities in the immune system not only hamper tumor immunosurveillance, but also limit the efficacy of cancer immunotherapy. Meanwhile, the results of recent studies allow the belief that one is on the edge of a real breakthrough in this promising direction in cancer therapy. The 2(nd) International Conference 'Cancer Immunotherapy and Immunomonitoring (CITIM)' was the second meeting in Eastern Europe to specifically focus on the issue of immune regulation in the tumor environment, cancer immunotherapy, and immunomonitoring of immunotherapeutic clinical trials. This CITIM Conference held in Budapest, Hungary, was comprised from 12 plenary sessions, Best Abstract Award session, Poster session, and four Keynote lectures. Outstanding presentations and numerous productive discussions summarized the current place of the field and opened new directions for improving monitoring and therapy for patients with cancer.

Defining the critical hurdles in cancer immunotherapy.

Scientific discoveries that provide strong evidence of antitumor effects in preclinical models often encounter significant delays before being tested in patients with cancer. While some of these delays have a scientific basis, others do not. We need to do better. Innovative strategies need to move into early stage clinical trials as quickly as it is safe, and if successful, these therapies should efficiently obtain regulatory approval and widespread clinical application. In late 2009 and 2010 the Society for Immunotherapy of Cancer (SITC), convened an "Immunotherapy Summit" with representatives from immunotherapy organizations representing Europe, Japan, China and North America to discuss collaborations to improve development and delivery of cancer immunotherapy. One of the concepts raised by SITC and defined as critical by all parties was the need to identify hurdles that impede effective translation of cancer immunotherapy. With consensus on these hurdles, international working groups could be developed to make recommendations vetted by the participating organizations. These recommendations could then be considered by regulatory bodies, governmental and private funding agencies, pharmaceutical companies and academic institutions to facilitate changes necessary to accelerate clinical translation of novel immune-based cancer therapies. The critical hurdles identified by representatives of the collaborating organizations, now organized as the World Immunotherapy Council, are presented and discussed in this report. Some of the identified hurdles impede all investigators; others hinder investigators only in certain regions or institutions or are more relevant to specific types of immunotherapy or first-in-humans studies. Each of these hurdles can significantly delay clinical translation of promising advances in immunotherapy yet if overcome, have the potential to improve outcomes of patients with cancer.

Conference Scene: Immunotherapy reaches new milestones in cancer eradication.

Biotherapy is widely considered as the fourth treatment modality for patients with cancer, and uses the constantly increasing knowledge in molecular biology, cell biology and immunology. Biotherapy uses naturally occurring biological molecules (e.g., cytokines and antibodies) or works by the manipulation of normal biological mechanisms (controlling or inhibiting tumor growth). Important achievements in anticancer drug development are immunotherapeutic strategies recently approved by the US FDA as well as clinical data of the cancer patients treated in clinical trials. There is a need to expand these novel cancer immunotherapeutic modalities for cancer patients all over the world. To meet that goal, it is essential to spread the information, to summarize the new clinical data and to draw the conclusions from the clinical and preclinical investigations. These frontline tasks can be well advanced by organizing international conferences in this domain in less scientifically developed countries, with a significant tumor burden statistics. Therefore, special efforts were done to organize the 2nd International Cancer Immunotherapy and Immunomonitoring Conference (CITIM-2011) in Hungary.

Pioneering the trail of cancer immunotherapy.

The annual conference of the International Society for Biological Therapy of Cancer (iSBTc), recently renamed the Society for Immunotherapy of Cancer (SITC), provides clinicians and scientists with a uniquely broad yet focused view of the growing field of tumor immunology and cancer immunotherapy. In this time of exciting new developments in cancer immunotherapy, it is critically important to effectively guide new immune-based advances from bench to bedside to improve outcomes of patients with cancer. iSBTc/SITC with its dedicated leadership (past and present) provides this guidance through its Annual Meeting and associated programs. This year the society celebrated its 25th Anniversary in Hyatt Regency Washington on Capitol Hill (Washington DC, USA), with educational offerings from 30 September to 4 October 2010.

15th International Conference on Human Antibodies and Hybridomas. 14-16 April 2010, Tiara Park Atlantico Hotel, Porto, Portugal.

Antibodies and antibody conjugates are currently one of the largest classes of new drug entities under development. These versatile molecules are being investigated for the treatment of many pathological conditions, such as cancer and infectious, inflammatory and autoimmune diseases. Antibodies can exert biological effects as naked antibodies by themselves, or can be used as delivery agents conjugated with various drugs (e.g., immunoconjugates) and as tools of multistep targeting. Site-specific delivery of therapeutic agents has been the ultimate goal of the pharmaceutical industry, as it has the potential to maximize drug efficiency while minimizing side effects. Antibodies have much potential for this objective. Thus, it is useful to summarize some of the main strategies currently being employed for the development of these diverse therapeutic molecules and to highlight the recent novelties in the field. These goals were the focus of the 15th International Conference on Human Antibodies and Hybridomas, held during 14-16 April 2010 in Porto, Portugal.

Turning laboratory findings into therapy: a marathon goal that has to be reached.

The mission of translational research involves difficult tasks to be accomplished for its ultimate goal, i.e., the introduction of novel, effective therapeutic strategies in the clinic to diminish human suffering and cure life-threatening diseases. Translational research (also referred to as translational medicine) facilitates the translation of investment in biomedical research into successful medical treatment. This includes the transfer of diagnostic and therapeutic advances by proving their efficacy in large evidence-based trials. Through the study of humans novel insights about disease are brought back to the laboratory to identify new, observation-based strategies. This "two-way road" ("bench to bedside and bedside to bench") process includes formulating guidelines for drug development and principles for new therapeutic strategies; initiating clinical investigations that provide the biological basis for new therapies, and related clinical trials; defining therapeutic targets and clinical endpoints. It requires a systematic approach beginning with specimen sampling, patient data collection, laboratory investigations, data analysis, preclinical testing, clinical trials, treatment efficacy monitoring, and finally the evaluation of therapeutic result. The marathon well symbolizes the enormous efforts undertaken by clinicians, scientists, regulators, ethicists, patient advocates, drug developers, and others, coordinately attempting to overcome obstacles along this road toward the final "marathon goal in medicine".

Antibody phage display: overview of a powerful technology that has quickly translated to the clinic.

Antibody-based immunologic reagents are useful for identifying, isolating, or eliminating cells with particular characteristics related to different diseases. Phage display is a highly valuable technique for antibody selection related to this purpose. In brief, a diverse group of antibody genes prepared from a patient or generated in vitro are inserted into a phagemid vector or the phage genome so that when the protein is expressed, it becomes anchored on the surface of the phage by fusion to a coat protein. A diverse library of recombinant antibodies is generated in this way and can then be exposed or panned on the antigen of interest, typically, this being a molecule associated with a particular pathological condition. Phage that carry proteins or peptides bind preferentially to the target and can thus be isolated from the library. The viruses that are recovered in this way also carry the gene for the binding moiety facilitating its over-expression or manipulation. Recent reviews highlight key milestones in the development of antibody libraries and their screening by phage display, and the impact of these technologies on drug discovery seems assured.

Intravenous immunoglobulin-based immunotherapy: an arsenal of possibilities for patients and science.

The use of intravenous immunoglobulin (IVIG) concentrated from pooled healthy donors' plasma has gained increasing popularity. IVIG therapy has become important as a replacement therapy in primary and acquired humoral immunodeficiencies, and it has been extended to autoimmune, neurodegenerative and inflammatory conditions and transplantation therapy. Recurrent pregnancy failure and cancer are rather new platforms, where IVIG has shown its beneficial effects. This manuscript is focused on these two off-labelled usages. The immunomodulatory mechanisms of IVIG therapy appear as a coordinated orchestration of different functions, resulting in a synergistic effect. Treatment monitoring and detailed molecular analyses reveal how such treatments may interfere with disease pathogenesis. These finding may foster the development of novel therapeutic and/or preventive strategies. Studying this field with bidirectional bench-to-bedside and bedside-to-bench approaches fit well into 'the two-way road' paradigm of translational medicine.

Cocktails of human anti-cancer antibodies show a synergistic effect in nude mouse tumor xenografts.

A panel of four natural human monoclonal IgG antibodies derived from B lymphocytes isolated from regional draining lymph nodes of cancer patients has been developed and characterized. The four human antibodies are termed, RM1, RM2, RM3, and RM4. The immunoreactivity of this panel of four human antibodies is restricted to tumor cells. Individually, these human MAbs show tumor targeting and are effective in inhibiting tumor growth in nude mouse xenograft models. When used in combination the antibodies show an additive effect in slowing down the progression of tumors in xenograft models suggesting that cocktails of antibodies may be useful in the clinic.

Alloimmune and autoimmune background in recurrent pregnancy loss - successful immunotherapy by intravenous immunoglobulin.

PROBLEM: Immunotherapies [leukocyte immunization, intravenous immunoglobulin (IVIG)] introduced to treat women with recurrent spontaneous abortions (RSA) have still controversial results in most clinical trials. A selection of these patients would be advantageous for higher efficacy. METHOD OF STUDY: A complex immunological panel assay was offered to patients with reproductive failure without any other known cause. We focused here on the cellular immunological parameters. RESULTS: High cytotoxic T lymphocyte precursor frequency and cell-mediated cytotoxic activity and a rather high natural killer cell activity were found in alloimmune RSA patients. Thirty-two patients were investigated by immunological assays and in 78% of the women an alloimmune background could be defined. The efficacy of IVIG treatment was 96% in this group. CONCLUSIONS: The novel cellular immunological assays proved to be favourable for the indication of RSA patients and showed the usefulness of this selection process for effective immunotherapy.

[Intravenous immunoglobulin treatment of recurrent spontaneous abortion with immunopathological background]. / Az immunpatológiai hátteru habituális vetélés kezelése terhesség alatti intravénás immunglobulinnal.

INTRODUCTION: Recurrent spontaneous abortion (RSA) is diagnosed if three or more spontaneous abortions follow each other typically in the first trimester. The root cause of miscarriages often can not be found. A significant proportion of this unexplained RSA cases may be caused by immunopathological failure. AIM: A multicentric clinical study started in 2000 to introduce an immunological screening protocol for patients suffering in idiopathic habitual abortion, and to use immunotherapy for their treatment if immunological background was defined. METHOD: The general checkup of the patients was managed based upon a detailed protocol, with which non-immunopathological reasons for RSA were excluded. The unexplained RSA cases underwent an immunological checkup including cellular and humoral immunological, immunogenetic and autoimmune examinations. Based upon these parameters, the immunopathological background of RSA was certified or excluded. In the confirmed immunopathological cases intravenous immunoglobulin (IVIG) therapy was applied during their next pregnancy, with continuous monitoring of the immunological parameters. RESULTS: 120 patients with RSA were examined, and 32 of them got IVIG therapy during their next pregnancy. In 72% of cases (23/32) IVIG treatment for RSA with immunopathological alloimmune background was successful, with the outcome of healthy newborn. Of the 9 unsuccessful cases, in 6 patients subsequently additional non-immunopathological reasons were diagnosed for their RSA. IVIG treatment of patients with clear alloimmune background was successful in 88.5% (23/26). CONCLUSION: Results show that immunopathological checkup and immunotherapy is a useful treatment in the modern medicine for the patients with unexplained RSA. However the success of this method depends on the adherence of the checkup protocol, because unsuccessful therapy of non-clear cases can reduce the efficiency.

Novel ganglioside antigen identified by B cells in human medullary breast carcinomas: the proof of principle concerning the tumor-infiltrating B lymphocytes.

The potential tumor-recognizing capacity of B cells infiltrating human breast carcinoma is an important aspect of breast cancer biology. As an experimental system, we used human medullary breast carcinoma because of its heavy B lymphocytic infiltration paralleled to a relatively better prognosis. Ig-rearranged V region V(H)-J(H), Vkappa-Jkappa, and Vlambda-Jlambda genes, amplified by RT-PCR of the infiltrating B cells, were cloned, sequenced, and subjected to a comparative DNA analysis. A combinatorial single-chain variable fragment Ab minilibrary was constructed out of randomly selected V(H) and Vkappa clones and tested for binding activity. Our data analysis revealed that some of the V(H)-J(H), Vkappa-Jkappa, and Vlambda-Jlambda region sequences were being assigned to clusters with oligoclonal predominance, while other characteristics of the Ab repertoire were defined also. A tumor-restricted binder clone could be selected out of the single-chain variable fragment kappa minilibrary tested against membrane fractions of primary breast tumor cells and tumor cell lines, the V(H) of which proved to be the overexpressed V(H)3-1 cluster. The specific binding was confirmed by FACS analysis with primary breast carcinoma cells and MDA-MB 231 cell line. ELISA and thin layer chromatography dot-blot experiments showed this target Ag to be a ganglioside D3 (GD3). Our results are a proof of principle about the capacity of B cells infiltrating breast carcinomas to reveal key cancer-related Ags, such as the GD3. GD3-specific Abs may influence tumor cell progression and could be used for further development of diagnostic and/or therapeutic purposes.

Tumor-infiltrating B cell immunoglobulin variable region gene usage in invasive ductal breast carcinoma.

A major focus of tumor immunology is to reveal the potential role and capacity of immunocompetent cells found in different solid tumor tissues. The most abundant infiltrating cells (TIL), the T lymphocytes have been investigated in details concerning T-cell receptor usage and specificity. However, B cells have hardly been investigated in this respect, although high cellular B-cell infiltration has been correlated with improved patients' survival in some breast carcinomas. This led to our objectives to study variable region gene usage of the tumor-infiltrating B cells in different breast carcinoma types. By defining the immunoglobulin repertoire of the tumor-infiltrating B lymphocytes in the most common invasive ductal carcinoma (IDC) of the breast we compared it to the rare medullary breast carcinoma (MBC). After phenotyping infiltrating ductal carcinomas, B cells were obtained from tumor tissue by microdissection technique. Numerous rearranged TIL-B immunoglobulin heavy chain V genes (VH) were amplified, cloned, sequenced, and comparatively analyzed. Some characteristics were found for both breast carcinoma types. The immunoglobulins produced by TIL-B in ductal carcinoma are highly matured and oligoclonal. We conclude that Ig variable region gene usage reveals similar and distinguishable characteristics of TIL-B immunoglobulin repertoires, which are representative of the nature of the immune responses in invasive ductal and medullary breast carcinomas.