What is Preclinical Immunogenicity Assessment?

By Michael Greenwood, M.Sc.Reviewed by Sophia Coveney

Assessing the immunogenicity, the ability of a foreign substance to elicit an immune response, of any drug or therapeutic is essential to assessing efficacy and toxicity at the early stages of drug discovery.

Image Credit: Sakurra/Shutterstock.com

However, traditional small molecule drugs tend not to elicit a significant response compared with biologics such as the proteins and monoclonal antibodies that are now deployed clinically. Protein therapeutics represent an increasingly utilized class of medicines that are used to treat a wide range of autoimmune diseases, genetic disorders, cancers, and infectious diseases.

However, the introduction of exogenous proteins into the body very frequently elicits an immune response, resulting in the production of antibodies against the therapeutic protein that neutralizes them and may potentially induce further health issues.

Vaccines, in contrast, aim to induce an immune response, allowing the body to develop a large number of high-affinity antibodies against the invader without being excessive and damaging in this process themselves for optimum efficacy.

How is an immune response developed?

Following capture and uptake by dendritic or B cells the therapeutic is degraded, and a subset of its constituent peptides loaded onto Human leukocyte antigens (HLA), cell-surface proteins involved in self-identification and immune modulation. The loaded HLAs are presented to T cells that go on to target and eliminate the therapeutic from the body.

Both proteins that are entirely foreign to humans and those that are usually endogenous are known to elicit immune responses when introduced, even with no apparent difference in the structural or chemical properties of the molecule. For example, Bococizumab is a humanized monoclonal antibody that targets PCSK9, an enzyme involved in lipoprotein homeostasis, and was administered to patients to lower LDL cholesterol levels. However, the drug was discontinued in 2016, in part due to the large portion of patients developing a significant immune response towards the protein, notably lowering its efficacy.

Proteins containing peptides with strong HLA affinity have been shown to increase the probability of an immune response towards the protein from which the peptide originated. In silico and in vitro measurements of peptide-HLA affinity have some predictive power in estimating the magnitude of immune response likely to be elicited by a protein, based on the examination of random peptide sequences from the proteins.

Unfortunately, in silico, in vitro, and animal models developed thus far are in their infancy, and are overall poorly predictive of immunogenicity. Assessment is usually performed by screening against a battery of antibodies, where more detailed assays are subsequently performed to assess whether any positive antibodies actually inhibit protein function upon binding.

How is immunogenicity determined?

Initial screening is usually done by enzyme-linked immunosorbent assay (ELISA), where known antibodies are bound to a solid substrate and a sample is added. Colorimetric or fluorimetric indicators of bonding between the antibodies and components within the sample can be easily visually identified.

The overall immune response to a foreign substance can also be assessed by measuring total antibodies produced over baseline, possibly using quantitative analytical methods such as inductively-coupled plasma optical emission spectroscopy/mass spectrometry.

Total immunoglobulin A/G/M concentration in serum in animal models is highly indicative of the magnitude and stage of immune response, with IgM being produced mainly at the initial stages of infection and IgG being the most abundant later in cases of pathogenic infection.

Immune response towards vaccines, or their vectors, can also be assessed at the preclinical stage by similar methods. The titer of antibodies specific to the target can be measured by quantitative methods, along with the presence of immune response-associated signaling molecules such as interferon.

It is determined whether the identified antibodies are neutralizing by other biological assays that may track a number of parameters, such as cell proliferation, mRNA or cytokine production, or monitor some other downstream effect.

Serum samples from humans or animals, with or without added antibodies, are added to the cells in concurrence with the therapeutic protein or vaccine as a control, allowing the effect of the therapeutic to be identified exclusively.

For example, if the usual response of the cell to the therapeutic is enhanced proliferation, then the presence of the neutralizing antibody will lessen the rate. Differences in animal models are often attributed to additional antibody clearance facilitated by other mechanisms, which may be assessed to some extent in vitro by including serum samples without added antibodies from the relevant patient. Drugs or biomolecules that inactivate the therapeutic protein can then be identified. This assay is most useful when applied to therapeutics with a low or difficult-to-measure pharmacologic effect in animal models, as the resulting magnitude of the effect of interfering components is so subtle in these cases.

Other factors of immunogenicity

Other factors that contribute towards an immune response include the aggregation, oxidation, and conformational state of the therapeutic. The development of immunogenicity is also highly dependent on the frequency and size of the dose received.

Single injections generally do not generate antibodies quickly enough to affect the protein therapeutic within the window of time before ordinarily expected clearance, though repeated exposure produces antibodies in increasing concentrations with increasing binding affinity towards the therapeutic.

Proteins can also be directly produced within host cells by delivering mRNA that instructs for the production of almost any desired protein, as in some of the recently developed COVID-19 vaccines that code for the production of the SARS-CoV-2 spike protein.

mRNA generally does not induce an immune response as significant as whole-protein delivery, though often requires a delivery vehicle or vector that is associated with an immune response, particularly in use in vaccines that intend to elicit such a response where additional immunostimulating adjuvants are often added.