Development of a new antibody-drug conjugate against CD71, INA03, for the treatment of leukemia

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Bratti et al. INA03: A Potent Transferrin-Competitive Antibody–Drug Conjugate against CD71 for Safer Acute Leukemia Treatment, Molecular Cancer Therapeutics, 2024

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The safety of INA03, a novel antibody-drug conjugate targeting CD71 (TFRC), was evaluated in a double humanized mouse model developed by genOway, providing support for the development of new cancer therapies.

CD71: a promising target for anti-cancer therapies

Antibody-drug conjugates (ADC) present a promising strategy as cancer therapy¹. ADCs consist of a cytotoxic drug linked through a cleavable linker to a humanized antibody with high specificity to its target. Once the ADC binds to its specific antigen, it is internalized by the target cell where the linker is cleaved, releasing the cytotoxic drug and promoting killing of the cancer cells. Cancer cells are known to upregulate the expression of CD71, a receptor for transferrin that is encoded by the TFRC gene and is an essential protein for the transport of iron in plasma. CD71 is present in all cells, albeit at different levels, and plays an important role in iron uptake. Cancer cells are known to upregulate the expression of CD71 to answer the high iron demands during their proliferation². Due to this increased expression on their surface, the CD71 receptor has become a biomarker with potential use for anti-cancer therapies.

In a recent study³, Bratti and colleagues developed a new antibody-drug conjugate, named INA03, to treat leukemia. INA03 consists of a humanized antibody that specifically targets CD71⁴ expressed at increased levels in highly proliferative cells, and competes with transferrin. This antibody is coupled with an antimitotic drug, MMAE, which inhibits cell division⁵, leading to cell death. Characterization of INA03 revealed a high affinity and specificity for human CD71. CD71 was efficiently internalized, allowing the release of MMAE into the cells, and validating this strategy as an effective method to deliver MMAE to cancer cells.

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INA03's cytotoxic activity is specific to cells expressing human CD71

The authors then assessed whether internalization of MMAE led to the desired effect of killing cancer cells. Cell viability was measured in HL60 and U937 cells upon incubation with INA03 in the presence or absence of transferrin. It was observed that INA03 successfully induces cell toxicity, but is less efficient at killing cells in the presence of transferrin. Cytotoxicity following incubation with INA03 was only observed in cells expressing human CD71, and not in cells expressing murine CD71, suggesting that cytotoxicity is induced through the specific targeting of human CD71 at high density.Activity of INA03 was also tested in vivo. To validate the function of INA03 as a cytotoxic agent that could reduce tumour burden, immunodeficient mice were inoculated with THP-1, HL60 and TL-Om1 cell lines and treated with INA03 (see figure below, panels B, C, D). Remarkably, INA03 treatment efficiently increases mouse survival and tumor depletion (panel G). However, this is dependent on the leukemia model used (panels B, C, D), combined with the drug concentration and mode of administration.

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Figure 4 in the paper: (B) Survival curve of THP-1 xenografted nude mice injected i.p. with INA03 at the indicated concentrations or PBS (1 injection every 4 days, for 4 times). (C) Survival curve of HL60 xenografted nude mice injected i.v. with INA03 at the indicated concentrations or PBS. (D) Survival curve of TL-Om1 xenografted nude mice injected i.v. with INA03 at the indicated concentrations or PBS. (G) Bioluminescence images of NSG mice bearing orthotopic xenografted HL60-Luc cells. INA03 at 0.5 mg/kg injected i.v. (1 injection every 4 days, 8 times) Red arrow represents day of HL60-Luc cell injection. i.p.: intraperitoneal, i.v.: intravenous. Adapted from Bratti et al., 2024³.

To study the effect of INA03 in a human physiological context with a high level of predictability, mouse models containing either humanized CD71 (hCD71), humanized transferrin (hTf), or both (double Knockin of hCD71/hTf, 2KI), were developed by genOway. This was achieved by knocking in human Tf and CD71 and disrupting the murine genes. In hCD71/hTf mice (2KI) INA03 was not fully internalized, showing the competition of human transferrin with INA03. To assess in vivo pharmacokinetics and toxicity of INA03, WT and 2KI mice were treated with different doses of INA03. INA03 clearance was faster in 2KI mice than in WT mice, validating both the specificity of INA03 to hCD71 as well as the use of the 2KI mouse model for pharmacokinetic studies. When administered at a high dose (15 mg/kg), INA03 did not cause toxicity or weight loss in mice, nor did it induce a strong pro-inflammatory response, as confirmed by an unaltered release of IFNγ, TNFα, IL-1β and IL-10.

This study provides an insight into the value of using CD71 (TFRC) as a target for cancer therapy, and the advantages of employing ADCs. INA03 delivers a drug that specifically kills cancer cells expressing abundant CD71, but it also competes with transferrin, depriving cancer cells of iron uptake. Importantly, the development of the hCD71/hTf mouse model allows for the validation of new therapies against human leukemia and lymphoma with high specificity and high predictability, and to assess the efficacy and toxicity of new chemotherapies.

A humanized TFRC model  is available off the -shelf at genOway, designer and provider of multiple preclinical models in several research areas, including immuno-oncology, metabolism, cardiovascular diseases and neuroscience.

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References

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3. Bratti, M. et al. INA03, a potent transferrin-competitive antibody-drug conjugate against CD71, for a safer acute leukemia treatment. Mol. Cancer Ther. (2024) doi:10.1158/1535-7163.MCT-23-0548.
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