Targeting CD83 in mantle cell lymphoma with anti-human CD83 antibody

Objectives: Effective antibody–drug conjugates (ADCs) provide potent targeted cancer therapies. CD83 is expressed on activated immune cells including B cells and is a therapeutic target for Hodgkin lymphoma. Our objective was to determine CD83 expression on non-Hodgkin lymphoma (NHL) and its therapeutic potential to treat mantle cell lymphoma (MCL) which is currently an incurable NHL. Methods: We analysed CD83 expression on MCL cell lines and the lymph node/bone marrow biopsies of MCL patients. We tested the killing effect of CD83 ADC in vitro and in an in vivo xenograft MCL mouse model. Results: CD83 is expressed on MCL, and its upregulation is correlated with the nuclear factor κB (NF-κB) activation. CD83 ADC kills MCL in vitro and in vivo. Doxorubicin and cyclophosphamide (CP), which are included in the current treatment regimen for MCL, enhance the NF-κB activity and increase CD83 expression on MCL cell lines. The combination of CD83 ADC with doxorubicin and CP has synergistic killing effect of MCL. Conclusion: This study provides evidence that a novel immunotherapeutic agent CD83 ADC, in combination with chemotherapy, has the potential to enhance the efficacy of current treatments for MCL

Novel Targets in Acute Myeloid Leukaemia

Background: Antibody based immunotherapies have revolutionised the treatment of haematological malignancies. Despite recent advances in Acute Myeloid Leukaemia (AML) most patients still have poor outcomes. Current surface targets in AML are not ideal and ongoing work is required to examine new antigens for meaningful clinical outcomes. Hypothesis: CD302 and CD300f have inherent properties that make them promising potential targets in AML. Preclinical work will establish these antigens as suitable targets in AML for further study. Methods: We looked at the distribution of CD302 on AML and Haematopoietic Stem and Progenitor Cells (HSPC) using flow cytometry from local patient cohorts and compared this data with AML gene expression profiling from public databases. The expression of CD302 from healthy organs was examined using PCR. The rate of internalisation of anti-CD302 antibodies was assessed using flow cytometry and fluorescent microscopy. The ability of unmodified antibodies to perform Antibody Dependant Cellular Cytotoxicity (ADCC) was examined. The impact of CD302 on AML cell migration was assessed using a transwell system. The influence of an anti-CD302 antibody upon AML cell line engraftment was tested in vivo. CD300f on AML and HSPC was analysed using flow cytometry from local patient cohorts and we compared the data to gene expression profiling from public databases. The distribution of CD300f isoforms across AML and HSPC was assessed by PCR and confirmed with TCGA RNA sequencing data. Alterations in antibody binding epitopes using multiple CD300f antibodies was assessed in AML cell lines as well as primary AML and HSPC. The cytotoxicity of an anti-CD300f based ADC was examined in vitro using cell lines. An anti-CD300f ADC was examined in vitro and in vivo against cell lines as well as primary AML and healthy HSPC. The rate of cytotoxicity and synergy of the ADC with Fludarabine was assessed in vitro with cell lines. Results: In a cohort of 33 AML patients, 88% were found to express CD302 on the surface of blasts and 80% on the surface of CD34+ CD38- population. Expression of CD302 was found on the surface of HSPC. A monoclonal antibody (mAb) targeting human CD302 was effective in mediating ADCC and was internalised, making it amenable to toxin conjugation. Targeting CD302 with this antibody limited in vivo engraftment of the leukaemic cell line HL-60 in NOD/SCID mice. While CD302 was expressed in a hepatic cell line, HepG2, this molecule was not detected on the surface of HepG2, nor could HepG2 be killed using a CD302 antibody-drug conjugate. CD300f antibodies bind to AML from 85% of patient samples. Transcriptomic analysis found that CD300f transcripts are expressed by healthy HSPC. Several CD300f protein isoforms exist as a result of alternative splicing. The extracellular region of CD300f can be present with or without the exon 4‐encoded sequence. This results in CD300f isoforms that are differentially bound by CD300f‐specific antibodies. Analysis of publicly available transcriptomic data indicated that CD34+ HSPC expressed fewer CD300f transcripts that expressed exon four compared to AML with monocytic differentiation. An anti-CD300f antibody, DCR‐2, to CD300f exposes a structural epitope recognized by a second CD300f mAb, UP‐D2. Analysis of a small cohort of AML cells revealed that this UP‐D2 conformational binding site could be induced in cells from AML patients with monocytic differentiation but not those from other AML or HSPC. CD300f is expressed evenly across HSPC subtypes. CD300f has equivalent transcription and protein expression as CD33 on AML. We have developed an anti-CD300f antibody which efficiently internalises into target cells and conjugated it with a PBD warhead that selectively depletes AML cell lines and AML Colony Forming Units (CFU) in vitro. CFU derived from healthy HSPC are depleted by the ADC. The ADC synergises with Fludarabine, which is often used in allogeneic Haematopoietic Stem Cell Transplant (allo-HSCT) conditioning. The ADC prolongs the survival of mice engrafted with human cell lines and depletes primary human AML engrafted with a single injection. In a humanised mouse model, a single injection of the ADC depletes CD34+ HSPC and CD34+ CD38- CD90+ HSC. Conclusions: CD302 is a potential target in AML. The hepatic expression may limit potential therapeutics but this could be mitigated as hepatocytes appear to express CD302 predominantly intracellularly, further work is required in this field. CD302 is expressed on HSPC and any future therapeutics would likely need to be part of a conditioning strategy. CD300f is a more promising target given the lack of non-haematopoietic expression. Certain isoforms or epitopes may be targeted to generate selective binding to AML with monocytic differentiation and avoid HSPC. An anti-CD300f ADC has promise as a targeted conditioning agent that may deplete residual AML and facilitate allo-HSCT

Is Hematopoietic Stem Cell Transplantation Required to Unleash the Full Potential of Immunotherapy in Acute Myeloid Leukemia?

From monoclonal antibodies (mAbs) to Chimeric Antigen Receptor (CAR) T cells, immunotherapies have enhanced the efficacy of treatments against B cell malignancies. The same has not been true for Acute Myeloid Leukemia (AML). Hematologic toxicity has limited the potential of modern immunotherapies for AML at preclinical and clinical levels. Gemtuzumab Ozogamicin has demonstrated hematologic toxicity, but the challenge of preserving normal hematopoiesis has become more apparent with the development of increasingly potent immunotherapies. To date, no single surface molecule has been identified that is able to differentiate AML from Hematopoietic Stem and Progenitor Cells (HSPC). Attempts have been made to spare hematopoiesis by targeting molecules expressed only on later myeloid progenitors as well as AML or using toxins that selectively kill AML over HSPC. Other strategies include targeting aberrantly expressed lymphoid molecules or only targeting monocyte-associated proteins in AML with monocytic differentiation. Recently, some groups have accepted that stem cell transplantation is required to access potent AML immunotherapy and envision it as a rescue to avoid severe hematologic toxicity. Whether it will ever be possible to differentiate AML from HSPC using surface molecules is unclear. Unless true specific AML surface targets are discovered, stem cell transplantation could be required to harness the true potential of immunotherapy in AML

Measurable Residual Disease in Acute Myeloid Leukemia Using Flow Cytometry: A Review of Where We Are and Where We Are Going

The detection of measurable residual disease (MRD) has become a key investigation that plays a role in the prognostication and management of several hematologic malignancies. Acute myeloid leukemia (AML) is the most common acute leukemia in adults and the role of MRD in AML is still emerging. Prognostic markers are complex, largely based upon genetic and cytogenetic aberrations. MRD is now being incorporated into prognostic models and is a powerful predictor of relapse. While PCR-based MRD methods are sensitive and specific, many patients do not have an identifiable molecular marker. Immunophenotypic MRD methods using multiparametric flow cytometry (MFC) are widely applicable, and are based on the identification of surface marker combinations that are present on leukemic cells but not normal hematopoietic cells. Current techniques include a “different from normal” and/or a “leukemia-associated immunophenotype” approach. Limitations of MFC-based MRD analyses include the lack of standardization, the reliance on a high-quality marrow aspirate, and variable sensitivity. Emerging techniques that look to improve the detection of leukemic cells use dimensional reduction analysis, incorporating more leukemia specific markers and identifying leukemic stem cells. This review will discuss current methods together with new and emerging techniques to determine the role of MFC MRD analysis

Examination of CD302 as a potential therapeutic target for acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most common form of adult acute leukemia with ~20,000 new cases yearly. The disease develops in people of all ages, but is more prominent in the elderly, who due to limited treatment options, have poor overall survival rates. Monoclonal antibodies (mAb) targeting specific cell surface molecules have proven to be safe and effective in different haematological malignancies. However, AML target molecules are currently limited so discovery of new targets would be highly beneficial to patients. We examined the C-type lectin receptor CD302 as a potential therapeutic target for AML due to its selective expression in myeloid immune populations. In a cohort of 33 AML patients with varied morphological and karyotypic classifications, 88% were found to express CD302 on the surface of blasts and 80% on the surface of CD34+ CD38- population enriched with leukemic stem cells. A mAb targeting human CD302 was effective in mediating antibody dependent cell cytotoxicity and was internalised, making it amenable to toxin conjugation. Targeting CD302 with antibody limited in vivo engraftment of the leukemic cell line HL-60 in NOD/SCID mice. While CD302 was expressed in a hepatic cell line, HepG2, this molecule was not detected on the surface of HepG2, nor could HepG2 be killed using a CD302 antibody-drug conjugate. Expression was however found on the surface of haematopoietic stem cells suggesting that targeting CD302 would be most effective prior to haematopoietic transplantation. These studies provide the foundation for examining CD302 as a potential therapeutic target for AML

CD83 is a new potential biomarker and therapeutic target for Hodgkin lymphoma

Chemotherapy and hematopoietic stem cell transplantation are effective treatments for most Hodgkin lymphoma patients, however there remains a need for better tumor-specific target therapy in Hodgkin lymphoma patients with refractory or relapsed disease. Herein, we demonstrate that membrane CD83 is a diagnostic and therapeutic target, highly expressed in Hodgkin lymphoma cell lines and Hodgkin and Reed-Sternberg cells in 29/35 (82.9%) Hodgkin lymphoma patient lymph node biopsies. CD83 from Hodgkin lymphoma tumor cells was able to trogocytose to surrounding T cells and, interestingly, the trogocytosing CD83+T cells expressed significantly more programmed death-1 compared to CD83–T cells. Hodgkin lymphoma tumor cells secreted soluble CD83 that inhibited T-cell proliferation, and anti-CD83 antibody partially reversed the inhibitory effect. High levels of soluble CD83 were detected in Hodgkin lymphoma patient sera, which returned to normal in patients who had good clinical responses to chemotherapy confirmed by positron emission tomography scans. We generated a human anti-human CD83 antibody, 3C12C, and its toxin monomethyl auristatin E conjugate, that killed CD83 positive Hodgkin lymphoma cells but not CD83 negative cells. The 3C12C antibody was tested in dose escalation studies in non-human primates. No toxicity was observed, but there was evidence of CD83 positive target cell depletion. These data establish CD83 as a potential biomarker and therapeutic target in Hodgkin lymphoma