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One of the great surprises of the biologics revolution has been the discovery that recombinant human proteins, including fully human monoclonals such as Humira, can cause immune responses when administered to immune-competent subjects. An even greater surprise, to some, is that tools are currently available to predict whether or not an antibody will be immunogenic in the clinic.

EpiVax has been providing access to our validated set of immunogenicity screening tools since 2002 – and – perhaps even more shocking, given the rather unscientific approaches that have confused the field of immunogenicity prediction in the past few years, we have excellent (published) record of validated predictions.

Previously, preclinical and clinical evaluations of the potential immunogenicity of biologics have been focused on humoral immune responses and as a result, the critical contribution of T cells to the development of anti-monoclonal antibodies (also known as anti-drug antibodies or ADA) has been somewhat overlooked.

More recent publication (see Koren et al., for example) have confirmed the role of effector T cells and begun to explore the role of regulatory T cells in the development of anti-drug antibodies. EpiVax has discovered key ‘regulatory’ T cell epitopes that are present in some, but not all monoclonal antibodies (see De Groot and Martin, Clinical Immunology May 2009, for more information, and see also De Groot A.S., et al, Activation of Natural Regulatory T cells by IgG Fc-derived Peptide “Tregitopes”. Blood, 2008,112: 3303.

The presence of these Treg epitopes in IgG has been validated in research conducted by EpiVax and five collaborating laboratories. Their presence or absence correlates with immune response in the clinic. In this very brief update, we take a look at two monoclonal antibodies, Rituximab and Ofatumumab, and we compare and contrast their predicted (and observed) immunogenicity based on T cell epitope and Tregitope content. Taking out our Immunogenicity Futuroscope, we give an A+ to the Ofatumumab antibody.

Background information

In studies conducted in autoimmune disease settings, 30-50% of patients have responded to Rituximab with the development of an anti-Ab (drug) response (ADA). Reportedly, ofatumumab has a potentially reduced immunogenicity compared with the other products due to it’s “human” origin. Reports from several studies are available on line but precise descriptions of Ofatumumab immunogenicity are not yet published:

In RA: “At 24 weeks, the patients’ immune responses to study medication (ofatumumab or placebo) were also evaluated by testing for the presence of human anti-human antibodies (ADAs).” All patients tested negative at 24 weeks. GlaxoSmithKline (GSK) and Genmab A/S announced these positive primary efficacy data (evaluated at 24 Weeks) at EULAR 2007, the Annual European Congress of Rheumatology (abstract number: OPO232) from a Phase II study of ofatumumab (HuMax-CD20®) in patients with rheumatoid arthritis (RA). Read more here.

In CLL: Ofatumumab was generally well tolerated by CLL patients in the study. The most frequently reported adverse events (those that occurred at a greater than 15% frequency) were: pyrexia, diarrhea, fatigue, cough, neutropenia, anemia and pneumonia. There were no unexpected safety findings. None of the 14 patients tested for human anti-human antibodies (HAHA) demonstrated their presence at 12 months. Read more here.

EpiVax analysis: Rituximab vs. Ofatumumab

In a previous analysis of Rituximab (Clinical Immunology, May 2009) we noted that there were no known Tregitopes (although 1 cluster in VH and 1 cluster in VL have a moderate homology with known Tregitopes). Since there are no true Tregitopes, the overall immunogenicity score for Rituximab is not adjusted; it is 4.12 for VH and 43.79 for VL. This is in the same range of scores for monoclonals that are currently in the clinic and known to be immunogenic (see De Groot and Martin analysis of Mabs in Clinical Immunology, May 2009). Therefore, we wished to determine whether a new monoclonal, Ofatumumab, also targeting CD20, would have reduced immunogenicity in the clinic.

The density of T-helper epitopes may explain differences in observed antibody responses to slightly different versions of the same recombinant human protein, such as that observed to chimeric and humanized monoclonal antibodies. The EpiVax “T cell epitope immunogenicity scale” allows the evaluation and comparison of protein sequences for immunogenicity based on T-cell epitope content.

Given the resulting “immunogenicity score” of a protein, and taking into consideration other determinants such as Tregitopes, as described above, it is possible to make an informed decision about the likelihood that a protein may provoke an immune response in the clinic.

In general, proteins having higher epitope densities tend to be more immunogenic while low-density proteins tend to be immunologically inert.


We obtained the Fab sequence of Ofatumumab from the RCSB Protein Database (ID 3GIZ), and performed a comparison to Rituximab. We then evaluated each protein for the density of T cell epitopes per amino acid.The approach has been described in detail in our publications, so we won’t go into great detail here.

Results and Conclusion:

Ofatumumab’s variable heavy chain contains four known Tregitopes, dramatically reducing its potential immunogenicity score from 48.14 to -55.43. Its variable light chain contains one Tregitope and two neo epitopes, reducing its EPX score from -12.89 to -17.79. It should be noted that the two neo epitopes share significant homology to known IgG1-derived Tregitopes.

Using our special immunogenicity futuroscope, we predict that Ofatumumab will be much less immunogenic that Rituximab in the clinic. EpiVax gives an A+ rating to this new monoclonal antibody and looks forward to clinical validation of our predictions, once again.