Abstract 5662: JNJ-67571244: A novel anti-CD33 C2 domain binding bispecific antibody with potent T cell redirection activity

CD33 is expressed in 90% of acute myeloid leukemia (AML) patients with expression in both blasts and leukemic stem cells. The extracellular portion of CD33 consists of a V and a C2 Ig-like domain. A recent study showed that a single nucleotide polymorphism (SNP) rs12459419 (C >T; Ala14Val in exon 2 of CD33) was present in ~50% of the Western AML population and is associated with preferential expression of an alternatively spliced CD33 isoform lacking exon2, resulting in the deletion of the CD33 V domain. Interestingly, several CD33-antibody-based therapies, including gemtuzumab ozogamicin (GO), the only approved anti-CD33 antibody drug conjugate for AML, bind and recognize the V domain of CD33. Recent data demonstrated that patients with SNP rs12459419 CC genotype receiving GO had a significantly lower risk of relapse and increased event-free survival compared to patients with the CT or TT genotypes. Given the data with GO, it is reasonable to hypothesize that the activity of other V binding CD33 antibodies maybe limited to a subset (~50%, rs12459419 CC genotype) of AML patients. On the other hand, since the C2 domain is shared by all CD33 isoforms in AML patients, we hypothesized that a C2 binding anti-CD33 antibody could target AML cells more broadly regardless of their SNP status. Additionally, we reasoned that targeting the membrane proximal C2 domain would be a beneficial strategy for a CD3 redirection bispecific antibody as targeting epitopes closer to the membrane have been reported to mediate efficient synapse formation between T cells and target cells leading to potent anti-tumor responses. We developed JNJ-67571244, a novel human bispecific antibody capable of binding to the C2 domain of CD33, and to CD3 to induce T-cell recruitment and tumor cell cytotoxicity independently of their SNP status. JNJ-67571244 specifically binds to CD33-expressing target cells and induces cytotoxicity of CD33+ AML cell lines in vitro at 48 hours (EC50 values: KG-1=0.168 nM, MOLM-13=0.131 nM, Kasumi-1=0.05 nM and OCI-AML3=0.183 nM) with concomitant T cell activation (EC50 values: KG-1=0.066 nM, MOLM-13=0.028 nM, Kasumi-1=0.043 nM and OCI-AML3=0.05 nM) along with cytokine release. In contrast, JNJ-67571244 was unable to kill CD33- cancer cell lines (CARNAVAL and KG-1 cells with a genetic deletion of CD33), demonstrating the specificity of the cytotoxicity. JNJ-67571244 demonstrated statistically significant anti-tumor activity in vivo in established disseminated and s