Preclinical Development Of ROR1 Peptide Based Vaccine With Activity Against Chronic Lymphocytic Leukemia In ROR1 Transgenic Mice

Abstract

Receptor tyrosine kinase-like orphan receptor (ROR1) is expressed on chronic lymphocytic leukemia (CLL) and other cancers, but not on normal post-partum tissues, except for a small subset of precursor B cells known as hematogones. Because of its restricted expression on cancer cells, ROR1 is an attractive target for developing novel anti-cancer therapies. In this study, we designed several different peptides, corresponding to distinct epitopes in the extracellular domain of ROR1. Each peptides was conjugated with keyhole-limpet hemocyanin (KLH) to generate a peptide-KLH vaccine, which we used to immunize C57BL/6 mice, first in complete Freund’s adjuvant (CFA) and then again 2 weeks later in incomplete Freund’s adjuvant (IFA). Two weeks following the second injection the sera from immunized mice were tested for binding activity for recombinant ROR1 protein via immunoblot analyses or ELISA or for cell-surface ROR1 by flow cytometry. We identified one peptide-KLH vaccine (R22-KLH) that induced high-titer anti-ROR1 antisera specific for ROR1-expressing cells that did not react with normal human or mouse tissues, which did not express this protein. The antisera generated by mice immunized with R22-KLH were capable of directing specific complement-dependent cytotoxicity (CDC) against neoplastic cells that expressed ROR1 (e.g. human CLL cells, EW36, or JeKo-1), but not against cells that did not express ROR1 (e.g. Jurkat). To study the capacity of R22-KLH to break self-tolerance, we immunized C57BL/6 mice transgenic for human ROR1 controlled by the immunoglobulin μ heavy-chain promoter/enhancer, designated as ROR1-Tg mice. Despite having B cells that express surface ROR1, such animals still were able to generate high-titer anti-ROR1 antisera comparable in binding and CDC activity and specificity as antisera generated in C57BL/6 mice. Production of such antisera was associated with reduced expression of ROR1 by the B cells of such ROR1-Tg mice. Cohorts of C57BL/6 mice or ROR1-Tg mice were immunized with R22-KLH or KLH and then challenged with intravenous injections of 3 × 105 or 2 × 104 ROR1-expressing primary CLL cells of double-transgenic ROR1xTCL1 C57BL/6 mice. Immunization with R22-KLH, but not KLH, significantly inhibited leukemia engraftment, as demonstrated by evaluation of the spleen and lymphoid tissues 4 weeks after adoptive transfer. To generate vaccines that might be suitable for clinical studies, we examined the relative immunogenicity of R22-KLH mixed with newly-developed agonists of toll-like receptor 4 (TLR-4; 1Z105) and TLR-7 (1V270). For this, we compared the immune responses of C57BL/6 mice or ROR1-Tg mice to R22-KLH administered s.c. in CFA/IFA to that of R22-KLH administered s.c. with 1Z105/1V270 in saline. Either C57BL/6 or ROR1-Tg mice generated higher titers of anti-ROR1 antibodies following immunization with R22-KLH in 1Z105/1V270 than did animals immunized with R22-KLH in CFA/IFA. Moreover, injection of R22-KLH in 1Z105/1V270 appeared better tolerated than injection of R22-KLH in CFA/IFA. These data indicate that immunization with R22-KLH can break tolerance to ROR1 and induce high-titer anti-ROR1 antisera. The immunogenicity of R22-KLH can be enhanced by injection with agonists of TLR4/7, obviating use of CFA/IFA. Pharmacology/toxicology studies are ongoing to determine the suitability of R22-KLH in 1Z105/1V270 for clinical trials involving patients with ROR1-expressing cancers. Disclosures: No relevant conflicts of interest to declare.

Spontaneous Immunity Against the Receptor Tyrosine Kinase ROR1 in Patients with Chronic Lymphocytic Leukemia

“…An induced antibody response against ROR1 also seemed to have clinical effects in vivo. ROR1 transgenic mice immunized with a ROR1 peptide developed high-titers of anti-ROR1 antibodies inhibiting engraftment of human ROR1 + primary CLL cells [ 11 ].…”

“…The antibodies inhibited Wnt5a dependent proliferation of CLL cells induced by Wnt5a. Immunization of ROR1 transgenic mice with ROR1 peptides induced anti-ROR1 antibodies, which inhibited engraftment of human ROR1 + CLL cells [ 11 ].…”

Dishevelled proteins are significantly upregulated in chronic lymphocytic leukaemia

“…Transgenic leukemic ROR1+ mice immunized with a ROR1 peptide produced high titers of anti-ROR1 antibodies which inhibited the engraftment of ROR1+ CLL cells [296] .…”

The ROR1 antibody-drug conjugate huXBR1-402-G5-PNU effectively targets ROR1+ leukemia

“…Targeting mechanisms include small molecule inhibitors, immunoliposomes, immunotoxins, bispecific antibodies, chimeric-antigen receptor modified (CAR)-T cells, ROR1 peptide vaccines, and monoclonal antibodies. [10][11][12][13][14][15][16][17][18][19][20] Early phase 1 clinical studies of cirmtuzumab, an anti-ROR1 monoclonal antibody (UC-961 clone), in patients with CLL showed that it was safe in patients without doselimiting toxicities. 18 Although cirmtuzumab failed to eradicate disease, this early trial established ROR1 as a safe therapeutic target.…”