GPR183 mediates the capacity of the novel CD47-CD19 bispecific antibody TG-1801 to heighten ublituximab-umbralisib (U2) anti-lymphoma activity
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Evaluation Summary:
Ribeiro M et al investigate the ability of a novel bispecific CD19-CD47 antibody to enhance the cell mediated killing mediated by existing drug combinations - anti-CD20 plus PIK3d/CK1E inhibitor. The novelty of this study is the restriction to CD19 positive lymphoma cells, thus potentially avoiding toxicity to non-lymphoma lineages, and the gene expression profiling to identify up regulation of GPR183 after combined treatment of CD19/47 plus CD20/PI3K/CK1E vs CD19/47 alone. Genetic and drug studies suggest that GPR183 is essential for the full activity of the triplet drug combination.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
Targeted therapies have considerably improved the survival rate of B-cell non-Hodgkin lymphoma (B-NHL) patients in the last decade; however, most subtypes remain incurable. TG-1801, a bispecific antibody that targets CD47 selectively on CD19+ B-cells, is under clinical evaluation in relapsed/refractory B-NHL patients either as a single-agent or in combination with ublituximab, a CD20 antibody, which is also being combined with the PI3Kδ/CK1e inhibitor, umbralisib (“U2”-regimen). In this study, we demonstrated that TG-1801 potentiates ublituximab-mediated antibody-dependent cell death (ADCC) and antibody-dependent cell phagocytosis (ADCP), leading to an additive anti-tumour effect of the TG-1801/U2 combination in B-NHL co-cultures. Accordingly, in a B-NHL xenotransplant model, the triplet achieved a 93% tumour growth inhibition, with 40% of the animals remaining tumour-free 35 days after the last dosing. Transcriptomic analysis further uncovered the upregulation of the G protein-coupled receptor, GPR183, as a crucial event associated with TG-1801/U2 synergism, while pharmacological blockade or genetic depletion of this factor impaired ADCP initiation, as well as cytoskeleton remodelling and cell migration, in B-NHL cultures exposed to the drug combination. Thus, our results set the preclinical rationale and support a combination strategy of TG-1801 with PI3Kδ- and CD20-targeting agents in patients with B-NHL.
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Evaluation Summary:
Ribeiro M et al investigate the ability of a novel bispecific CD19-CD47 antibody to enhance the cell mediated killing mediated by existing drug combinations - anti-CD20 plus PIK3d/CK1E inhibitor. The novelty of this study is the restriction to CD19 positive lymphoma cells, thus potentially avoiding toxicity to non-lymphoma lineages, and the gene expression profiling to identify up regulation of GPR183 after combined treatment of CD19/47 plus CD20/PI3K/CK1E vs CD19/47 alone. Genetic and drug studies suggest that GPR183 is essential for the full activity of the triplet drug combination.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the …
Evaluation Summary:
Ribeiro M et al investigate the ability of a novel bispecific CD19-CD47 antibody to enhance the cell mediated killing mediated by existing drug combinations - anti-CD20 plus PIK3d/CK1E inhibitor. The novelty of this study is the restriction to CD19 positive lymphoma cells, thus potentially avoiding toxicity to non-lymphoma lineages, and the gene expression profiling to identify up regulation of GPR183 after combined treatment of CD19/47 plus CD20/PI3K/CK1E vs CD19/47 alone. Genetic and drug studies suggest that GPR183 is essential for the full activity of the triplet drug combination.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This study focused on a bi-specific antibody, TG-1801, which binds to both CD19 and CD47. The overall objective of this study was to examine the anti-tumor efficacy of combining TG-1801 and U2-regimen (ublituximab and umbralisib) in B-cell non-Hodgkin Lymphoma as a triplet treatment, and to understand the underlying mechanisms of the synergism.
The authors demonstrated superior effects of the triplet therapy in inducing antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) of CD19-expressing B-cell lymphoma cells. Using a Raji tumor mouse model, the triplet treatment achieved significant tumor inhibition. The transcriptional analysis identified an upregulation of GPR183 in B-cell lymphoma cells upon triplet treatment but not in TG-1801 or U2 treatment. Knockdown …
Reviewer #1 (Public Review):
This study focused on a bi-specific antibody, TG-1801, which binds to both CD19 and CD47. The overall objective of this study was to examine the anti-tumor efficacy of combining TG-1801 and U2-regimen (ublituximab and umbralisib) in B-cell non-Hodgkin Lymphoma as a triplet treatment, and to understand the underlying mechanisms of the synergism.
The authors demonstrated superior effects of the triplet therapy in inducing antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) of CD19-expressing B-cell lymphoma cells. Using a Raji tumor mouse model, the triplet treatment achieved significant tumor inhibition. The transcriptional analysis identified an upregulation of GPR183 in B-cell lymphoma cells upon triplet treatment but not in TG-1801 or U2 treatment. Knockdown of GPR183 expression in Raji cells diminished ADCP and ADCC induced by TG-1801, U2 or the triplet treatment. The inhibition of cell migration and F-actin intensity by TG-1801, U2 or the triplet treatment were also reversed in the absence of GPR183.
The strengths include: appropriate models have been used for evaluating the efficacy of the combination treatment; the superior efficacy of triplet therapy has been demonstrated with both in vitro and in vivo experiments; GPR183 was identified as a critical mediator for the effects of the triplet therapy and its function was evaluated with loss-of-function models.
The weaknesses include: the rationale of how GPR183 was chosen from differentially expressed genes was not clearly interpreted; the role of GPR183 in mediating the effects of TG-1801/U2 combination therapy was inconclusive based on the data provided in the manuscript and was not adequately examined with appropriate models.
Overall, this interesting study supported a rational combination of TG-1801 and U2-regimen for B cell non-Hodgkin lymphoma treatment with preclinical models. However, mechanistically, the role of GPR183 in mediating the effects of the triplet treatment remained inconclusive.
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Reviewer #2 (Public Review):
The authors examine whether their CD19/47 bispecific can potentiate ADCC and ADCP mediated killing in preclinical lymphoma models.
The ability of CD47 blockade to synergize with an alternative CD20 (Rituximab) is already established in preclinical models (Choa et al. Cell 2010) and the combination has been deployed in patients (Advani et al NEJM 20018). The novelty of the current study CD47 blockade is now targeted exclusively to CD19 positive B cells . The rationale for this being that it might reduce toxicity to non-B lineages such as the erythroid lineage, since anemia was a significant toxicity in previous studies of CD47 blockade.
Using a single CD19 positive and single CD19 negative cell line the authors confirm CD19-dependent blockade of CD47 at a dose of 2ng/ml.
In vitro assays for phagocytosis in a …
Reviewer #2 (Public Review):
The authors examine whether their CD19/47 bispecific can potentiate ADCC and ADCP mediated killing in preclinical lymphoma models.
The ability of CD47 blockade to synergize with an alternative CD20 (Rituximab) is already established in preclinical models (Choa et al. Cell 2010) and the combination has been deployed in patients (Advani et al NEJM 20018). The novelty of the current study CD47 blockade is now targeted exclusively to CD19 positive B cells . The rationale for this being that it might reduce toxicity to non-B lineages such as the erythroid lineage, since anemia was a significant toxicity in previous studies of CD47 blockade.
Using a single CD19 positive and single CD19 negative cell line the authors confirm CD19-dependent blockade of CD47 at a dose of 2ng/ml.
In vitro assays for phagocytosis in a panel of 5 lymphoma cell lines show increased phagocytosis in all cell lines treated with eat CD19/47 antibody. In a single cell line (Raji), phagocytosis is then increased further by the addition of the umbralisib/ublituximab combo (U2). The weakness here that it is not clear if the effect seen in Raji represents synergy or additive killing, and moreover phagocytosis compared to CD19/47 alone is only seen in one of five cell lines. ADCC assays are also shown but it is not clear from the methods section how these assays distinguish effector cell-mediated killing from direct apoptosis induction.
Xenografting of a single cell line (Raji) shows that tumor growth is suppressed by the CD19/47 and appears to be suppressed further by addition of U2. Some data is presented for percent of mice tumor-free after a further 35 days, but any difference seems not to achieve statistical significance.
Therefore the data presented in figure 1 provide only limited support for authors' conclusions that there is synergy between CD19/47 and U2, and the data that any potentiation of cell killing is all due to the induction of a cell-mediated response rather than apoptosis is not compelling.
Using gene expression, the authors then identify upregulation of GPR183 in cells treated with TG108 plus U2 versus TG108 alone. They show that GPR183 loss abolishes M1 macrophage infiltration in Raji organoids and abolishes phagocytosis, the latter effect being partly recapitulated by pharmacological inhibition. From the current experimental design, it is not clear if GPR183 is induced by the CD20 engagement or by the PI3K/CK1E inhibition, or whether it even requires the presence of CD47 engagement at all. However, the implication of this pathway in regulating ADCP is important and will spur further investigation.
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