In early 2022, a collaborative investigation from Australian and Austrian cancer and research institutions reported exciting results from a preclinical experiment in murine models with myeloproliferative neoplasms.1 The promising data, published in EMBO Reports, have the potential to modify how myeloproliferative disorders (MPD), including myelofibrosis (MF), are treated in the future.
To date, MF is considered an “undruggable” condition that is acquired due to somatic mutations that result in expansion of the myeloid cellular lineage and activation of hematopoietic signaling pathways. Mutations to calreticulin (CALR) are the most common type, leading to the formation of an abnormal peptide sequence that stimulates the pathways.
The research team hypothesized that this may serve as a targetable neoepitope that, when blocked, may halt the abnormal signaling cascades associated with the development of MPDs. The positive results published by the research team are discussed below and may drive early clinical trials in humans as early as 2023.
Dr. Daniel Thomas, who leads the Myeloid Metabolism Laboratory at the South Australian Health and Medical Research Institute and is a part of the discovery team, described the finding as “serendipitous.”2
Targeting Neoepitopes
More than two decades ago, it was proposed that cancer cells express neoepitopes as a result of underlying somatic or passenger mutations. Neoepitopes are peptides that arise from somatic mutations and are recognized as different from self. Dendritic cells or other antigen-presenting cells present these nonself antigens to T cells or B cells to elicit a specific immune response.
In the case of cancer, the neoepitope is expressed on the surface of the tumor cell itself. Cancer immunotherapy aims to fight cancer by training the immune system to recognize these tumor-specific antigens.3 The recently discussed possibility of cancer vaccines becoming mainstream in the coming years is based on similar principles.
Read more about myelofibrosis
The neoepitope target under investigation for MPDs is associated with mutations to CALR. In primary myelofibrosis (PMF), CALR mutations are the second most common genetic aberration and are present in 70% of non-JAK2V617F and non-MPL types. CALR mutations in PMF are usually insertions or deletions that almost always occur in exon 9 and are classified as type 1 (most frequent) and type 2 (second most frequent). The type 1 mutation is a 52 base pair (bp) deletion and type 2 is a 5bp insertion, both on exon 9.1
The result of the mutation is the translation of an abnormal peptide that is thought to activate the thrombopoietin receptor (TpoR). The underlying mechanism is not understood but is thought to involve glycan-binding sites, the N-terminal chaperone domain, and the novel C-terminal tail of the mutant protein.
One important consequence of the mutation is the loss of the KDEL sequence—an endoplasmic reticulum retention signal. Without it, the abnormal mutant CALR (mutCALR) peptide is passively secreted across the cell membrane and is detectable in cultured cell supernatants.1 Evidence from a prior study suggests that TpoR activation occurs after cell surface exposure, which led the researchers to believe that an intracellularly acting agent can be developed to block this interaction.1,4
Monoclonal Antibody 4D7
A novel therapeutic agent against mutCALR would need to possess two 2 properties. Firstly, it ideally should work against both type 1 and type 2 mutations. Additionally, it should spare normal hematopoiesis.1
Read about the pathophysiology of myelofibrosis
The typical mutations to CALR lead to a loss of the negative-charged C-terminal calcium-binding domain. The researchers engineered a peptide that corresponded to the C-terminal mutCALR neoepitope with the intent to produce a murine monoclonal antibody against it. Several candidates were developed and ultimately 4D7 was selected based on its strong positivity during immunoblotting.1
4D7 Blocks TPO-Independent Signaling
Two cytokine-independent cell lines, TF-1 TpoR CALRdel61 and TF-1 TpoR CALRdel5, were developed and cultured to test the activity of 4D7 on cellular proliferation. Another culture of TF-1 cells without CALR mutations and that express TpoR was also developed because it was important to assess if 4D7 would have an effect on normal cells. As hoped, 4D7 did not inhibit growth in the control batch.
The takeaway here is that 4D7 does not inhibit proliferation through TpoR inhibition. In TF-1 TpoR CALRdel61 and TF-1 TpoR CALRdel5 cells, however, a concentration-dependent suppression of cellular proliferation was observed after 48 hours. Interestingly, in TF-1 TpoR CALRdel61 and TF-1 TpoR CALRdel5 cells that did not possess TpoR, no inhibition was observed.1
Normally, activation/phosphorylation of TpoR results in constitutive STAT and ERK phosphorylation. Proof that 4D7 functions through TPO-independent blockade was confirmed by the observation that phospho-STAT1/3/5 and phospho-ERK are downregulated in TF-1 cells expressing TpoR alone only after TPO withdrawal. However, in 4D7-treated TF-1 TpoR CALRdel61 and TF-1 TpoR CALRdel5 cells, blockade of factor-independent phospho-STAT1/3/5 and phospho-ERK is observed after incubation with 4D7. The mechanism by which 4D7 inhibits cellular proliferation is by inhibiting the formation of the mutCALR-TPO complex, which prevents activation of TPO and downstream signaling.1
The effect of 4D7 was not observed in factor-independent cells with JAK2V617F or PTPN11E76K mutations, suggesting that 4D7 is mutation-specific.1
4D7 Inhibits TPO-independent Megakaryocyte Formation
Clonal proliferation of abnormal megakaryocytes is a major characteristic of the pathophysiology of PMF. The effect of 4D7 on TPO-independent megakaryocyte differentiation in liquid culture and TPO-independent megakaryocyte colony formation on a collagen-based medium was assessed to determine if 4D7 suppressed CALR-mutant megakaryocytes.1
The results were positive in both experiments. For TPO-independent megakaryocyte differentiation in liquid culture, a mean decrease of 55% was observed across CALR-mutant samples after 4D7 treatment (P< 0.0001). In cells with the type 1 mutation, 80% displayed at least 50% inhibition when treated with 4D7. TPO-independent megakaryocyte colony formation on a collagen-based medium yielded similar results with a 62% reduction in the absolute numbers of primary TPO-independent megakaryocyte colonies treated with 4D7 in multiple samples (P< 0.0001).1
4D7 May Solve the Ruxolitinib Problem
There is a very short list of treatment options for PMF and the limitations of some medications put a proverbial handcuff on the treating physician since there are not many options when one drug fails. In some circumstances, these limitations may leave patients completely out of options. For example, ruxolitinib (Jakafi®) is an oral, reversible JAK1 and JAK2 inhibitor that is approved for the treatment of PMF, among other diseases, in adults. Some patients require dose increases as their disease advances; however, a major limitation of ruxolitinib is that it is associated with cytopenias at high doses, especially in patients with CALR mutations (vs JAK2 mutations).5
Read more about treatment of myelofibrosis
The researchers developed a ruxolitinib-resistant culture of TF-1 TpoR CALRdel61 cells and treated them with 4D7 to assess outcomes. To the research team’s relief, a strong inhibitory activity on cells that were resistant to ruxolitinib was observed in liquid culture at 96 hours. Colony formation was also strongly inhibited. The outcome of this experiment has exciting implications because it means that an immunotherapeutic approach with 4D7 may have clinical utility in mutCALR patients who develop resistance to ruxolitinib treatment. This may be a game-changer. Additionally, in vivo experiments demonstrated survival in mice with TF-1 TpoR CALRdel61 bone marrow engraftment that were ruxolitinib-resistant.1
In Vivo Experiments With 4D7
It was important to determine whether 4D7 is capable of inhibiting CALR-dependent proliferation in vivo. Two distinct cell-line xenograft models were developed for this purpose. The first was a bone marrow engraftment model to measure mutant CALR-dependent proliferation of TF-1 TpoR CALRdel61 cells in the bone marrow microenvironment.1 The second was a chloroma model (a neoplastic variant that involves the formation of cancerous blood cells via extramedullary hematopoiesis) by administering a subcutaneous injection of TF-1 TpoR CALRdel61 cells into the flanks of NSG mice, which mimics extramedullary hematopoiesis.1,6 The measured outcomes in this experiment were survival of both xenograft models, pharmacokinetic profile, and tumor growth, as fibrosis was not possible to evaluate.1
In the marrow engraftment model, 4D7 treatment resulted in significantly prolonged survival and showed a favorable pharmacokinetic profile. In the chloroma model, tumor growth was significantly slowed at 353 mm3 vs 3317 mm3 in control mice (P =0.0008).1
Where Will This Journey Take Us?
4D7 appears to be a promising candidate as a novel treatment option for MPDs including PMF, polycythemia vera, and essential thrombocytopenia. Dr. Thomas himself said in an interview that he thought the results were a mistake. He and the research team set out to prove a proof-of-concept and wound up with results that Dr. Thomas described as “too good to be true.”2
There are crucial tests that 4D7 must pass to live up to its hype. Firstly, it is critical to assess whether 4D7 is indeed not toxic to normal cells. Additionally, the results from this preclinical study leave a few unanswered questions. For example, is 4D7 efficacious in patients with heterozygous mutations? The effect on fibrosis could not be evaluated in the xenograft subjects; this would be an important outcome in clinical trials, especially if fibrosis can be reversed.
Dr. Thomas believes that 4D7 could begin enrolling patients for in-human trials within a year and, to his knowledge, scores of patients are eager to participate.
References
1. Tvorogov D, Thompson-Peach CAL, Foßelteder J, et al. Targeting human CALR-mutated MPN progenitors with a neoepitope-directed monoclonal antibody. EMBO Rep. 2022;23(4):e52904. doi:10.15252/embr.202152904
2. An antibody for myelofibrosis – “that’s a true discovery”. Leukaemia Foundation. Published July 13, 2022. Accessed December 26, 2022.
3. Brennick CA, George MM, Corwin WL, Srivastava PK, Ebrahimi-Nik H. Neoepitopes as cancer immunotherapy targets: key challenges and opportunities. Immunotherapy. 2017;9(4):361-371. doi:10.2217/imt-2016-0146
4. How J, Hobbs GS, Mullally A. Mutant calreticulin in myeloproliferative neoplasms. Blood. 2019;134(25):2242-2248. doi:10.1182/blood.2019000622
5. Jakafi. Package insert. Incyte; 2021. Accessed December 28, 2022.
6. Chloroma. National Cancer Institute. Accessed December 28, 2022.
