Patients with Chronic Lymphocyte Leukemia (CLL) Have Naturally Occurring Antibodies Against the Receptor Tyrosine Kinase (ROR1)

The receptor tyrosine kinase ROR1 is a tumor-associated antigen which is highly expressed by leukemic cells from patients with chronic lymphocytic leukemia (CLL) and in a variety of other hematologic malignancies. ROR1 is not expressed in normal adult tissues with the exception for adipose cells and a subset of normal B cells during early ontogeny. ROR1 has important functions during embryogenesis. Thus ROR1 is considered an oncofetal antigen (Fakuda T et al. PNAS, 105, 3047, 2008).

As ROR1 in adults represents a neo-antigen, we analysed spontaneously induced humoral immune response against ROR1 in CLL patients with non-progressive and progressive disease.

Antibodies against ROR1 was analysed in 23 CLL patients with non-progressive (n=15) or progressive (n=8) diseases, as well as in 13 healthy donors by ELISA and Western blot, using a recombinant ROR1 protein. The recombinant protein contained 101 amino acids of the KNG domain of the extracellular ROR1 part equipped with a GST tag. Immunoprecipitated ROR1 from CLL cells was also used as an antigen to further define the presence of anti ROR1 antibodies. Highly purified IgG and IgM from sera of CLL patients as well as healthy donors were used in flowcytometry for staining of CLL cells, expressing the ROR1 molecule. Regulatory T cells frequency (CD4+CD25+FoxP3+ and CD8+CD25+FoxP3+) was determined by flowcytometry. The presence of soluble ROR1 molecule was also investigated in 9 CLL patients. To detect soluble ROR1 from 9 CLL patients, serum was passed through HiTrap protein G, A and IgM columns to delete the pre existing IgM and IgG antibodies. The effluent sera were concentrated by Amicon Ultra 50000 Dalton tubes and concentrated serum from each patient was analysed by Western blotting to detect ROR1 molecules, using goat and rat anti ROR1 antibodies.

In 3 out of the 23 CLL patients anti-ROR1 antibodies were detected by ELISA and Western blot using the recombinant ROR1 protein (containing only the KNG domain), all these 3 patients had non-progressive disease. Using immunoprecipitated ROR1 protein from CLL cells as antigen, revealed the presence of anti ROR1 antibodies in all non-progressive CLL patients (15/15) by Western blot. Antibodies were also detected in patients with progressive disease (5/8) but at a lower concentration, compared to non-progressive patients. Flowcytometry staining of ROR1 expressing CLL cells using whole sera of patients and healthy donors as well as the purified IgG and IgM fractions, revealed the presence of both IgG and IgM antibodies from CLL patients reacting with ROR1 expressing CLL cells. Both IgM and IgG antibodies were detected in patients with non-progressive disease, but in progressive CLL patients only IgM antibodies were found. None of the sera of healthy donors and the purified IgG and IgM antibodies showed reactivity with CLL cells.

Analysis of the frequency of regulatory T cells revealed a significantly higher frequency of CD4+CD25+FoxP3+ cells in CLL patients compared to healthy donors. A higher frequency of these cells was observed in progressive compared to non-progressive CLL patients, but the differences were not statistically significant. Soluble ROR1 molecules of 105 and 130 KDa molecular weights were detected in sera of CLL patients (n=9) by goat (polyclonal) and rat (monoclonal) anti ROR1 antibodies using Western blot.

Our results showed that anti ROR1 antibodies are present in the sera of CLL patients, particularly in those with non-progressive disease as compared to progressive disease. These antibodies were both of IgM and IgG isotypes. The main source of soluble ROR1 molecules is probably apoptotic or necrotic cells and may contribute to the induction of a humoral anti-ROR1 response in addition to lysed tumor cells taken up by dendritic cells. Together with our previous results, exploring the presence of spontaneous T cell immunity in CLL patients, the current results confirm that ROR1 may be an immunodominant antigen in CLL patients. Active immunotherapy using ROR1 as an antigen might be an interesting approach to test in the clinic.

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