The Velocimmune Mice platform is a unique technology used for producing fully human monoclonal antibodies. It involves the precise replacement of the mouse heavy and light antibody variable region genes, with the corresponding human genes. Integral to the platform’s main functionality is training the mouse constant and regulatory regions to make a fully functional humoral immune system antibody response.
The Platform: Velocimmune Mice
One key element of this bispecifics platform includes the ‘universal light chain’, or ULC mice. These are velocimmune mice that possess the full complement of the human heavy chain variable region genes, but only a single pre rearranged human light chain. These mice produce fully human antibodies that all use the same light chain and thus obviate the issues of light chain mispairing. To make biospecific antibodies, Regeneron generates pairs of antibodies with the desired properties in their ULC mice to then express the biospecific antibody. This occurs in three main stages. Firstly, the heavy chain must be co-expressed from the first antibody, then the heavy chain from the second antibody, and finally the common light chain in the same CHO cell (Chinese hamster ovary cell).
To facilitate isolation of the biospecific antibody from the parental antibodies, the Velocimmune Mice platform incorporates a substitution from human IgG3 into one of the light chains. A heavy chain is chosen, and substitution disrupts binding a protein A. As a result, the parental conventional antibodies will either bind too weakly or too tightly to protein A. However, the device-specific antibody binds just right and can be purified over protein A. Consequently, the resulting bispecific antibody retains a native antibody format, with characteristics similar to a PK format. An advantage to using this platform is that the engineered bispecific possesses stability and has no unnatural mutations. Additionally, it can be manufactured efficiently at scale using conventional methods.
Case Study 1: Biparatopic Antibody
The first case study was a biparatopic ADC that targets the MET receptor. MET is a well-studied target for cancer therapy and refers to a canonical receptor tyrosine kinase where ligand-dependent signalling is mediated by receptor dimerisation. When mutated, MET serves as an oncogenic driver of lung and other cancers. Common mutations involve gene amplification and exon 14 skip mutations that disrupt normal ubiquitin-mediated degradation of the receptor. Blocking MET signalling is a validated approach to treatment. However, complete responses to therapy are not frequent, as almost all tumours go on to develop resistance. To overcome this, Regeneron developed the biparatopic antibody as one treatment strategy and then conjugated it to a potent cytotoxic agent for added efficacy.
Findings: The immunised ULC mice generated a sizeable panel of non-competing ULC antibodies that could be combined to form biparatopic antibodies. These biospecific antibodies showed unique properties relative to the parental antibodies, and one, named REGN5093, had the exact therapeutic properties desired. REGN5093’s potential to inhibit ligand-independent met signalling and downstream signalling was then tested. In the tumour bearing mice, REGN5093 localises effectively to the tumour as compared to the isotype control. Regeneron found that the level of uptake steadily built up over the course of four to six days and was not blocked at the higher dose of antibody. In humanised mice, there was significant uptake in the liver, being maximal on day zero and then diminishing over time. This was presumably as the tracer is metabolised and excreted. In contrast to the tumour, the liver uptake was largely blocked at the higher dose of the antibody.
Case Study 2: HER2 ADCs
Regeneron then switched to HER2 as a second case study to illustrate the more general applicability of their platform. HER2 is a member of the EGFR family of receptor tyrosine kinases, and it is overexpressed in approximately 15% of breast cancers and a smaller number of other cancers. HER2 expression is associated with very poor prognosis in the absence of treatment. Fortunately, the use of ADCs and small molecule TK-I means treatment for women with HER2 positive breast cancers has significantly improved. However, not all patients benefit from available therapies as complete responses are infrequent and recurrence is the norm. Additionally, targeted therapies remain unapproved for tumours with moderate to low HER2 expression.
The two approved HER2 ADCs are characterised by different therapeutic payloads mechanisms of cytotoxicity conjugation methods, payload release strategies, and ability to kill HER2 negative cells. Both employ trastuzumab as a targeting antibody and in Regeneron’s experiment, confocal microscopy was used to compare the relative rates of internalisation of trastuzumab and antibody to the prolactin receptor of the PRLR. The protein-coding gene or PRLR is employed as a model antigen due to its rapid and constitutive turnover.
Findings: Internalisation of the PRLR antibody was evident by its rapid disappearance from the surface and accumulation intracellularly. Trastuzumab largely remained on the cell surface, quantitatively about 90% of the PRLR antibody was internalised within 60 minutes, whereas minimal internalisation of trastuzumab was observed.
Analysis: Trafficking, Binding, and Toxicity
While REGN5093 was rather unique in its pharmacologic properties for HER2, Regeneron identified several HER2 by HER2 antibodies that significantly enhanced trafficking of trastuzumab and parental antibodies. The lead biparatropic antibody showed picomolar binding to HER2, comprising of Fab arms that did not cross compete with each other or with trastuzumab. This promoted rapid degradation of HER2-relative to trastuzumab, used alone or in combination with pertuzumab.
The sited toxicity of the ADC against cancer cells tracks very nicely with the expression of HER2. A 3mg/kg dose of the REGN5093 biparatopic ADC gave more effective tumour control than the 10 mg/kg dose of an in-house version of trastuzumab. But the differences were not so profound. This was potentially due to how these cells expressed very high levels of HER2 one and a half million copies per cell. Bigger differences were observed in the version of in HER2 or trastuzumab that had a dose of 10 mg/kg. This tumour was largely refractory to 10 mg/kg of trastuzumab but was highly sensitive to 3 mg/kg of our biparatopic ADC. The potential advantages of the biparatopic ADC became more apparent against such tumours with a 10 mg/kg dose of trastuzumab. One had very modest activity, even despite quite robust HER2 expression. In contrast, the 3 mg/kg dose of HER2-by-HER2 biparatopic ADC provided a complete tumour eradication of both tumours, as did a 1 mg/kg dose.
Regeneron was successfully able to show how biparatopic antibodies can fundamentally alter cellular traffic universe sceptre. It plans to continue to study the structural basis for such findings, along with examining the delivery of additional drug classes against a broader range of cancerous and normal cells. Regeneron also aims to test the tolerability of their platform in non-human primates, to assess how to best maximise anti-tumour activity, while minimising toxicity to normal tissues and maintaining favourable therapeutic index.