In general, in the first step of this PCR, a set of forward primers binding to the 5-end of the framework (FR) 1 of human variable domains, thereby adding the 3-portion of a universal leader peptide to the 5-end of the amplicon, was paired with a reverse primer specific to the rat constant domain expressed in H2L2 mice. as an ADC. (A) Supernatants from sorted single-cell cellular clones of the (GK) and (MK) libraries (GK = VH sequences cloned from IgGs, MK = VH sequences cloned from IgMs based Rabbit Polyclonal to CADM2 on different reverse primers used) derived from mice 1357, 1359, and 1363 were functionally screened for IgG levels and hROR2-ECD-Twin-Strep binding by ELISA in a single-well measurement. Normalized hROR2 binding was expressed as the ratio of OD = 490 nm hROR2-ECD-Twin-Strep binding and OD = 490 nm IgG expression for all clones from the different libraries and is shown as box-plots. (B) All supernatants were also assessed for potency as an ADC using a secondary ADC assay. For this, EMT6-hROR2 cells were incubated with clonal supernatant without normalization for IgG levels for 30 min, before addition of an anti-human Fc coupled via a cleavable linker to PNU-159682. Viable cells were quantified following a 3 days incubation using a luminescence-based cell viability assay. Lower luminescence values in the box-plots indicate more potent killing. Image_4.JPEG (337K) GUID:?2EEB119D-CA6A-4685-B819-F553ECA1CA84 Figure S5: Validation of killing potency of selected clones from functional ADC screening. Twelve clonal L11 supernatants with potent killing and four supernatants with poor killing CEP-18770 (Delanzomib) (GK-1C6, GK-1G6, MK-3E5, and MK-3A11) were CEP-18770 (Delanzomib) selected for testing in a secondary ADC assay using a range of defined concentrations to confirm CEP-18770 (Delanzomib) their cell killing potency. To do so, IgG levels of the supernatants were quantified by ELISA and IgG concentration in all supernatants was adjusted to the lowest expressor. EMT6-hROR2 cells were incubated with 2-fold serial dilutions of these normalized clonal supernatants for 30 min, followed by the addition of an anti-human Fc coupled via a cleavable linker to PNU-159682. After a 3 days incubation, viable cells were quantified using a luminescence-based cell viability assay. (A) Viability of the EMT6-hROR2 cells plotted in arbitrary units (a.u.) of luminescence on the y-axis as a function of the IgG concentration in the supernatants on the x-axis. Clonal supernatants that had potent or poor cell killing potency in the initial functional ADC screening are shown in black or gray, respectively. (B) shows luminescence values that had been determined in the one-well secondary ADC assay during functional ADC screening compared to the IC50 values which were determined in the secondary ADC assay using serial dilutions of normalized supernatants for the same clonal supernatants. IC50 values were calculated using a four-parameter curve fitting model in GraphPad Prism. n/a indicates IC50 values that could not be calculated due to a lack of killing. Image_5.JPEG (1.1M) GUID:?91B0C4B8-D9BF-4D1D-BA46-9247253A0768 Figure S6: SPR sensorgrams of anti-hROR2 antibodies. Affinities were measured by multi-cycle SPR on a Biacore T200 instrument (GE Healthcare). Antibodies were captured by Protein A or G immobilized on a CM5 sensor chip, followed by the addition of hROR2-ECD-Twin-Strep used as 2-fold serial dilutions ranging from 40 to 2.5 nM. KD values as a measure of binding CEP-18770 (Delanzomib) affinity are indicated. Image_6.JPEG (1.2M) GUID:?ED8EF759-7770-45B0-BB29-F60D155C9E86 Table S1: Germline V gene usage of identified anti-hROR2-clonotypes. The closest human germline V gene sequences of heavy (HC) and light chain (LC) were identified using IgBLAST. Image_7.JPEG (884K) GUID:?1ACE59CC-B159-4132-BE3B-33DF4E7F5368 Table S2: cell killing by anti-hROR2-ADCs. IC50 values (ng/ml) reported here represent the IC50 values of the hROR2-specific ADCs tested for their cell killing activity on hROR2-negative L363 and hROR2-high EMT6-hROR2 cell lines in Figure ?Figure7.7. IC50 values were calculated from the mean of two replicates using a four-parameter curve fitting model in GraphPad Prism. Image_8.JPEG (1.0M) GUID:?1F2F1014-822F-4770-8305-7F40C6F78AF7 Abstract Receptor tyrosine kinase-like orphan receptor 2 (ROR2) has been identified as a highly relevant tumor-associated antigen in a variety of cancer indications of high unmet medical need, including renal cell carcinoma and osteosarcoma, making it an attractive target for targeted cancer therapy. Here, we describe the discovery of fully human ROR2-specific antibodies and potent antibody drug conjugates (ADCs) derived thereof by combining antibody discovery from immune libraries of human immunoglobulin transgenic animals using the Transpo-mAb mammalian cell-based IgG display platform with functional screening for internalizing antibodies using a secondary ADC assay. The discovery strategy entailed immunization of transgenic mice with the cancer antigen ROR2, harboring transgenic IgH and IgL chain gene loci with limited number of fully human V, D, and J gene segments. This was followed by recovering antibody repertoires from the immunized animals, expressing and screening them as full-length human IgG libraries by transposon-mediated display in progenitor B CEP-18770 (Delanzomib) lymphocytes (Transpo-mAb Display) for ROR2 binding. Individual cellular Transpo-mAb.