Searching for key immune markers
Our researchers are focussed on discovering key immune markers and biological processes which will provide new diagnostic and therapeutic products for improving patient care.
Supervisors: Dr Xinsheng Ju and A/Prof Georgina Clark
The Dendritic Cell Research Group at the ANZAC Research Institute has forged a strong reputation in fundamental human dendritic cell research. Using its extensive clinical links and commercial collaborations, it is developing diagnostic and therapeutic antibodies for novel targets with application in clinical transplantation and the treatment of haematological and other malignancies.
Prostate cancer is the most common cancer in Australian men and new therapies are needed. We can train the immune system to fight cancer by vaccinating patients using specialised antigen presenting white blood cells, called dendritic cells. We have developed novel monoclonal antibodies together with practical methods to isolate these cells from patients. We can then present prostate cancer targets to the patient’s own immune system so that it will attack their cancer.
To be successful DC immune therapy must overcome practical hurdles associated with: 1) maintenance of DC function, i.e. their ability to migrate to and initiate immune responses in secondary lymphoid organs and 2) the optimal choice and delivery of prostate cancer tumour associated antigens (TAA) that addresses immunologically relevant human leucocyte antigen (HLA) differences amongst individuals to provide a therapy applicable to all patients.
We have developed methods, using our human-chimeric (h-) CMRF-56 monoclonal antibody (mAb) to isolate DC from patients that stimulate responses that kill cancer cells. We are developing a novel method of loading TAA into DC through using in vitro transcribed mRNA. This permits presentation of multiple antigens to T cells and allows patient-specific presentation of TAA peptides to the immune system. This will be compared to other loading methods such as DNA or peptide loading. We will optimise our dendritic cell vaccine by assessing it function in combination with different immune releasing “checkpoint inhibitor” therapeutics. This project aims to capitalize on these fundamental advances to obtain innovative preclinical data to support a clinical trial for our dendritic cell vaccine in prostate cancer.
This project will involve:
- Preclinical testing of clinically relevant adjuvants on primary human dendritic cell preparations to examine their effects on activation, cytokine production, migration and induction of T cell responses. (Primary human cell culture, flow cytometry, ELISA)
- Assessment of different combinations of TAA to load into CMRF56+ DC in order to achieve optimal prostate cancer responses. (Primary human cell culture, animal handling, flow cytometry)
- Assess anti-cancer immune responses induced by mRNA-loaded primary human dendritic cells in combination with different checkpoint inhibitors. (Primary Cell culture, Flow cytometry, immunofluorescence, cytotoxicity assays)
This project will provide the student with experience in different aspects of human immunology in a clinically relevant translational research environment that is being used to direct the development of clinical trials of DC mediated immune therapy in Sydney.
- Fromm, P. D., et al . (2016). CMRF-56(+) blood dendritic cells loaded with mRNA induce effective antigen-specific cytotoxic T-lymphocyte responses. Oncoimmunology. 5: e1168555.
Supervisor: A/Prof Georgina Clark and Dr Pablo Silveira
Dendritic Cell Research at the ANZAC Research Institute has forged a strong reputation in fundamental human dendritic cell research. Using its extensive clinical links and commercial collaborations, it is developing diagnostic and therapeutic antibodies for novel targets with application in clinical transplantation and the treatment of haematological and other malignancies.
The CD300 family of molecules, first described by our group consists of 7 closely related cell surface immunoregulatory molecules(1). The molecules are type I transmembrane glycoproteins that act as phospholipid binding molecules. Six members of the family are expressed by subpopulations of leucocytes and we are interested in developing therapeutic antibodies to these molecules to use as immune regulators. One member of this family, CD300e, is expressed primarily on monocytes(2). Its restricted expression makes it an attractive target for potential therapeutic manipulation of monocyte responses. However, there are few reagents available to study this molecule and so there is still much to learn about its function(3).
To investigate the biology of CD300e in vivo, we have generated CD300e-deficient (knock-out (KO)) mice. Our studies to date show that whilst the haematopoietic lineages are normal in number and frequency, monocyte/macrophage populations generated from the bone marrow of these mice have an enhanced inflammatory phenotype. The honours project will contribute to studies investigating the role of CD300e in tumour biology by performing comparisons of the ability of tumors to engraft CD300e KO and control wild type (WT) mice. The student will learn several laboratory skills including flow cytometry, real-time PCR, cell culture, isolation of lymphoid populations through magnetic- and fluorescence-activated cell sorting and optimisation of mouse tumour models assessed using a bioimager. As we are a strong translational laboratory, there are also options to collaborate on investigating CD300e biology in humans.
- 1. Clark GJ, Ju X, Tate C, Hart DN. The CD300 family of molecules are evolutionarily significant regulators of leukocyte functions. Trends Immunol. 2009;30(5):209-17.
- Clark GJ, Jamriska L, Rao M, Hart DN. Monocytes immunoselected via the novel monocyte specific molecule, CD300e, differentiate into active migratory dendritic cells. J Immunother. 2007;30(3):303-11.
- Gasiorowski RE, Ju X, Hart DN, Clark GJ. CD300 molecule regulation of human dendritic cell functions. Immunology letters. 2012.
Supervisor: Dr Pablo Silveira
Allogeneic haematopoietic cell transplantation (alloHCT) is the only curative therapy for many types of blood cancers (e.g. leukemia and lymphoma). The procedure involves exchanging a patient’s immune system with that of haematopoietic cells from a distinct donor. Before this is done, the recipient must undergo intensive regimens including radiation and chemotherapy to reduce the cancer and accept the graft. T cells within the donor graft have the capacity to eradicate the remaining malignant cells within patients in a process known as the Graft versus Tumour (GVT) response, adding to the effectiveness of the treatment. However, alloHCT recipients are at risk of developing Graft versus Host Disease (GVHD), a serious complication where donor T cells attack the host tissues. Our laboratory is interested in developing new therapeutics which alleviate GVHD while retaining the GVT effect, thus increasing the safety of this procedure.
In the past, alloHCT could not be used in the elderly or infirm due to the intensive radiation and chemotherapy regimens used to prepare patients for the procedure (known as myeloablative conditioning (MAC)). The introduction of reduced intensity conditioning (RIC) regimens that use greatly reduced levels of radiation with chemotherapy has increased access to this life saving procedure. Although RIC alloHCT patients still develop GVHD, it has become apparent that the kinetics and mechanisms underlying its development differs from patients receiving MAC alloHCT. To better understand how GVHD develops in the context of RIC and optimize potential treatments for these patients, new animal models are required.
Our group has developed a new mouse model of RIC alloHCT which recapitulates many of the features of human RIC alloHCT, including development of GVHD . RIC alloHCT recipient mice develop GVHD with slower kinetics and exhibit targeting of different organs (e.g. bone marrow) compared to MAC recipient mice. We have recently obtained luciferase transgenic mice which can be used as donors in this model. Due to their ability to bioluminesce, we can track how donor immune cells spread in recipient tissues in vivo using a Whole Body Bioimager .
The aims of this project are to:
[i] Compare immune cell infiltration of target tissues when GVHD develops over time in the context of RIC rather than MAC using a Bioimager.
[ii] Use flow cytometry and mass cytometry techniques to examine differences in activation markers on immune cells in RIC recipients that develop lethal GVHD compared to those that do not and in tissues targeted by GVHD compared to those that are not.
[iii] Use confocal and mass immuno-histology techniques to examine positioning of donor pathogenic T cells and antigen presenting cells in tissues targeted by GVHD in the RIC model.
- Shahin, K. et al. Bone Marrow Graft-Versus-Host Disease in Major Histocompatibility Complex-Matched Murine Reduced-Intensity Allogeneic Hemopoietic Cell Transplantation. Transplantation. 101: 2695-704.
- Beilhack, A. et al. In vivo analyses of early events in acute graft-versus-host disease reveal sequential infiltration of T-cell subsets. Blood. 106: 1113-22.
Supervisors: Dr Pablo Silveira and A/Prof Georgina Clark
The Dendritic Cell Research Group at the ANZAC Research Institute has forged a strong reputation in fundamental human dendritic cell research. Using its extensive clinical links and commercial collaborations, it is developing diagnostic and therapeutic antibodies for novel targets with application in clinical transplantation and the treatment of haematological and other cancers.
Dendritic cells (DC) are specialized myeloid immune cells, which survey and capture antigens in their local tissue environment before transporting them to draining lymph nodes (LN). The cells express a range of surface receptors that guide their migration into and within specific sectors of lymphoid tissues, where they can interact and present antigents to lymphocytes to mount an appropriate adaptive immune response. Identifying and understanding the function of these “migratory” receptors is key to developing the next generation of improved immune therapies that will prevent infection or treat cancer.
The C-type lectin receptor family of molecules play important roles in the immune system through their ability to recognise conserved carbohydrate structures on foreign pathogens and altered structures on damaged cells. However, some members of the family have the ability to bind endogenous ligands and have been reported to mediate alternate functions such as cell adhesion, cell migration, or LN expansion and contraction. Our group has identified a new member of the C-type lectin receptor family, named DEC205 associated C-type Lectin-1 (DCL-1) or CD302. Our studies in humans and mice have shown that CD302 is highly expressed on dendritic cells, monocytes, macrophages, pointing to a significant role within the myeloid immune system. We have generated the first CD302 knockout mouse strain and shown that it has an important role in the migration of dendritic cells into the T cell areas of lymph nodes. The mechanisms by which it does so and the ligand/s bound by CD302 remain to be defined.
The aim of this project is to identify and characterize the DC migratory mechanisms controlled by CD302. The project will involve generating a new monoclonal antibody to mouse CD302 to examine rare DC populations expressing this molecule and how it changes with activation using flow cytometry. Confocal, intra-vital and mass cytometry will be used to compare DC migration within lymph nodes and other tissues when the cells can or cannot express CD302. Finally, we will examine the recently identified ligands for CD302 and how these are used to regulate migration. Experiments will involve using cell culture and mouse models. As we are a strong translational laboratory, discoveries of CD302 function in mouse DC will be investigated in humans. This is an opportunity to gain experience in many different immunological techniques and contribute significantly to the understanding of an important new C-type lectin receptor. We see CD302 as an attractive target for potential therapeutic manipulation of immune responses to cancers, autoimmune diseases or infection.
- Kato, M., S. Khan, E. d’Aniello, K. J. McDonald, and D. N. Hart. 2007. The novel endocytic and phagocytic C-Type lectin receptor DCL-1/CD302 on macrophages is colocalized with F-actin, suggesting a role in cell adhesion and migration. J Immunol 179:6052-6063.
- Kato, M., S. Khan, N. Gonzalez, B. P. O’Neill, K. J. McDonald, B. J. Cooper, N. Z. Angel, and D. N. Hart. 2003. Hodgkin’s lymphoma cell lines express a fusion protein encoded by intergenically spliced mRNA for the multilectin receptor DEC-205 (CD205) and a novel C-type lectin receptor DCL-1. J Biol Chem 278:34035-34041.
- Lo, T. H., Silveira, P. A., Fromm, P. D., Verma, N. D., Vu, P. A., Kupresanin, F., Adam, R., Kato, M., Cogger, V. C., Clark, G. J., and Hart, D. N. Characterization of the Expression and Function of the C-Type Lectin Receptor CD302 in Mice and Humans Reveals a Role in Dendritic Cell Migration. 2016. J Immunol. 197: 885-98.
THE DENDRITIC CELL SURFACE
We seek to understand the cell surface phenotype of the human blood dendritic cell (BDC) populations. There is still a paucity of mAbs to dendritic cell molecules that can be used to positively select the cells. A select few mAbs bind molecules that, in many cases, have unknown identity and function however they are useful in discriminating different BDC populations. The function of the molecules identified with DC specific mAbs remains unknown in many cases. Our pathway to understanding the BDC surface and the molecular landscape is to combine studies using human cells in vitro models or xenogeneic in vivo models with specific mouse models. We focus on molecules that we were the first to identify and target a number of key molecules and antibodies that we are investigating in depth.
- CD300 family of immune regulatory molecules
- CD302 ; the simplest C-type lectin
- The CMRF-44 antigen
- The CMRF-56 antigen
TARGETING NOVEL MOLECULES WITH NOVEL MONOCLONAL ANTIBODIES
Therapeutic mAbs are the fastest growing biological class. We are using our portfolio of novel mAbs to understand how each can be used as a therapeutic in the treatment of haematological cancers. Our mouse mAbs are being engineered into human chimeric mAbs, humanised or new mAbs selected from human phage libraries. We are focusing on two areas in particular.
- Acute graft versus host disease is a life threatening complication of haematopoietic cell transplantation. We have shown that targeting activated DC prior to transplant prevents aGVHD. We have developed a human anti-human mAb that can be used to deplete activated DC and we are continuing mechanistic studies to understand how this works. We are preparing to take the mAb to Phase I clinical trial to test its safety and efficacy. We are also considering ways to use this mAb as a therapeutic as an immune suppressive in solid organ transplantation and other haematological malignancies.
- We are investigating ways of treating acute myeloid leukemia with myeloid specific mAbs using antibody drug conjugates and other antibody derivatives. We have three molecules we are targeting to treat AML.
- Checkpoint inhibitors have been used with great success in oncology. However their success has been limited to a limited number of cancer types and a limited group of patients. Antibodies targeting other checkpoint inhibitors are also likely to be successful. We are pursuing the use of mAbs to inhibitory molecules as novel checkpoint inhibitors.
DC VACCINATION FOR CANCER IMMUNOTHERAPY
Using DC to re-educate the immune response to cancer has long been a holy grail Many clinical trials have been completed which whilst demonstrating safety have been disappointing in their efficacy. Most DC trials have used the monocyte derived DC (MoDC). One of the most successful trials used a DC preparation derived from blood cells. We have long argued that MoDC do not represent the gold standard DC and that a preparation of BDC will provide an enhanced DC vaccine.
- We are have developed a BDC purification platform using one of our novel mAbs, CMRF-56, that results in an effective DC preparation for DC vaccination. We are optimising these cells for vaccines to treat castrate resistant prostate cancer, glioblastoma multiforme, colorectal cancer and acute myeloid leukemia.
- In vivo targeting of antigen to DC will be a cost efficient therapy. Our novel DC mA target internalised molecules. We are using them to deliver antigen to DC in induce anti-tumour responses.
OUR CURRENT PhD STUDENTS PROJECTS
Our Current PhD Students are focusing on the following projects;
- CD300e; a novel monocyte marker involved in regulating inflammation
- Antigen delivery through novel DC activation molecules
- Therapeutic antibodies and their derivatives to treat AML
- Understanding the mechanism of action of anti-CD83 treatment to prevent GVHD
- Treatments for lymphomas
- Combining DC vaccination with checkpoint inhibitors for prostate cancer, glioma and AML