Yet, secondary FLS and YLL ADCs tended to outperform their primary ADC counterparts when target cell cytotoxicity was directly compared at equivalent log[nM] concentrations. antibodies are marketed for the treatment of patients with cancer. This antibody format generally requires accessory immune components, such as complement and natural killer cells, to engage the Fc region of a bound antibody and eliminate tumor cells through complement-dependent cytotoxicity (CDC) or antibody-dependent Choline Fenofibrate cell-mediated cytotoxicity (ADCC), respectively.1Select antibody molecules may also abrogate downstream signaling cascades that are important in cell growth by interacting with surface-exposed targets such as growth factor receptors.2However, many external factors govern the clinical success of this targeted approach, including issues such as solid tumor penetrance and the moderate-to-high antibody binding needed to effectively elicit anti-tumor immunologic accessory responses or signaling pathway disruptions.3Additionally, with respect to CDC and ADCC, distinct Fc receptor polymorphisms in patients have been shown to dictate the degree of responsiveness to an antibody-based strategy.4,5 Engineered molecules such as antibody-drug conjugates (ADC) represent a conceptual improvement over the use of unmodified antibody molecules. Instead of relying on external factors to mediate cancer cell destruction, the antibody itself delivers a cytotoxic payload (i.e., small molecule drug) to tumor cells following endocytosis.6Cytotoxic drugs are stably attached to the antibody molecule via a short linker sequence and are released during endosome/lysosome fusion pathways, subsequently disrupting microtubule polymerization or DNA replication.7Several ADCs (brentuximab vedotin, ado-trastuzumab emtansine) are currently marketed and have met with degrees of success in treating malignant disease, albeit with CFD1 a narrow therapeutic index.8-10Efforts are now underway to further improve clinical efficacy by optimizing ADC properties such as systemic stability, antibody-linker chemistry, off-target cytotoxicity, and drug potency.11 Tumor antigen (TA) selection (with a preferred internalization profile) represents an additional area for therapeutic index improvement within the ADC field, but the selection is Choline Fenofibrate limited because of the need for sufficient target availability on cancer lesions to deliver adequate drug payloads.6,11Of the more than 50 ADCs in clinical development, none binds a given peptide/human leukocyte antigen (HLA) expressed by tumor cells.8The peptide/HLA is a unique targeting moiety since it is expressed by nucleated cells within the body and can phenotypically distinguish normal and malignant cells.12Indeed, considerable work within the area of immunotherapy has incorporated vaccines and genetic engineering to route effector CD8+ T cells to engage and destroy tumor cells through distinct peptide/HLA profiles.13Therapeutic antibodies (designated T cell receptor [TCR]-like antibodies) can also be raised to specifically bind a given peptide/HLA complex, much like a TCR engages the peptide/HLA.14Our group and others have successfully developed high affinity TCR-like antibodies against several tumor targets across different HLA alleles and reported favorable anti-tumor properties and protection in preclinical models.15-17In an extension of this work, we provide here proof-of-principle that TCR-like antibodies effectively function as small molecule drug carriers, particularly when delivering payloads to cancer cells that disrupt DNA replication (via duocarmcyin analogs). The TCR-like ADCs Choline Fenofibrate under study Choline Fenofibrate were capable of eliciting destruction of breast and colorectal cancer lines and diminishing tumor cell growth at physiologically relevant peptide/HLA surface-bound levels (3502,000 copies/cell).18,19Specific TCR-like antibody and peptide/HLA interactions also modulated ADC killing potential, indicating that, regardless of drug conjugate potency, TCR-like antibody docking to defined regions of the peptide/HLA severely dampened ADC cytotoxicity. Taken together, these findings provide support for the continued preclinical development of TCR-like ADCs, with a long-term goal of clinical evaluation in patients with cancer. == Results == == TCR-like antibodies deliver an immunotoxin to cancer cells but target copy number affects killing efficacy == Our group has reported around the power of unmodified TCR-like antibodies to specifically bind peptide/HLA, mediate CDC/ADCCin vitro, and provide protection against tumorigenic growthin vivo.20,21Interestingly, TCR-like antibodies engage their specific target and become internalized over a short period of time,16,22but we have not previously focused on the killing potential of TCR-like antibodies conjugated to toxins or small molecule drugs. To initially assess cytotoxicity, the TCR-like antibodies YLL-Ab and FLS-Ab (Supplementary Table 1) were indirectly labeled with the ribosomal inhibiting protein saporin (see Materials and Methods) and incubated alongside breast (MDA-MB-231 (Fig. 1A)) and colon adenocarcinoma (SW620 (Fig. 1B)) cell lines, which express cognate peptide/HLA-A2 ligands (Fig. S1). Although saporin levels remained constant in treated wells, increasing concentrations of YLL-Ab and FLS-Ab enhanced tumor cell death, suggesting a direct correlation with killing efficacy and internalized TCR-like immunotoxin complexes and payload release. These data.