Novel Antibody Drug Conjugates Targeting Tumor-Associated Receptor Tyrosine Kinase ROR2 by Functional Screening of Fully Human Antibody Libraries Using Transpo-mAb Display on Progenitor B Cells

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 de novo 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 lymphocytes ("Transpo-mAb Display") for ROR2 binding. Individual cellular "Transpo-mAb" clones isolated by single cell sorting and capable of expressing membrane-bound as well as secreted human IgG were directly screened during antibody discovery, not only for high affinity binding to human ROR2, but also functionally as ADCs using a cytotoxicity assay with a secondary anti-human IgG-toxin-conjugate. Using this strategy, we identified and validated 12 fully human, monoclonal anti-human ROR2 antibodies with nanomolar affinities that are highly potent as ADCs and could be promising candidates for the therapy of human cancer. The screening for functional and internalizing antibodies during the early phase of antibody discovery demonstrates the utility of the mammalian cell-based Transpo-mAb Display platform to select for functional binders and as a powerful tool to improve the efficiency for the development of therapeutically relevant ADCs.

Keywords: ROR2; antibody discovery; antibody drug conjugate (ADC); functional screen; human IgG transgenic mice; human immune library; mammalian IgG cell display; transposition.

Figure 1

Graphic representation of the discovery strategy for novel, fully human mAbs and functional ADCs from immune libraries of Ig transgenic mice. H2L2 Ig transgenic mice (Harbor BioMed) capable of generating antibodies with completely human V_H and V_L regions were immunized with soluble human ROR2-ECD-Twin-Strep. Transpo-mAb compatible libraries were generated from MACS-enriched antigen-specific splenocytes and co-electroporated into L11 mouse pro-B cells (with a RAG2-knock-out background) employing Piggy-Bac transposase to allow for stable integration. Cell clones expressing antigen-specific antibodies were single-cell sorted by FACS. Supernatants of these single-cell clones were directly screened for antigen-binding by ELISA and their suitability as an ADC by a secondary, functional ADC-based cell killing assay. Potent ADC candidates were subjected to sequence recovery of variable domain coding regions by RT-PCR and validated following expression of the antibodies in HEK293T cells.

Figure 2

hROR2-specific serum titers in H2L2 transgenic and in C57BL/6 wild-type mice. Five H2L2 Ig transgenic mice (A) and 5 wild-type C57BL/6 mice (B) were immunized with soluble hROR2-ECD-Twin-Strep. Serum samples were collected 7 days prior to immunization as well as on days 7, 28, and 49 post-immunization, i.e., always 7 days following each immunization. Titers in serum against hROR2-ECD-Twin-Strep as well as an unrelated soluble antigen also containing the same Twin-Strep-tag (Nonspecific Antigen-Twin-Strep) to assess tag-related responses were analyzed by ELISA. Bar graphs show mean values with standard deviation indicated.

Figure 3

Isolation of hROR2-binders from cellular libraries by FACS. Transpo-mAb-expression libraries were constructed by amplification of coding regions for human V_H and V_L domains with human variable domain specific primers using cDNA isolated from hROR2-ECD-Twin-Strep-specific splenocytes and cloning into transposable vectors, thereby fusing the variable domains in frame to the coding regions of either HCγ1 or LCκ constant domains. By use of Cγ1 or Cμ reverse primers, V_H domain encoding libraries have been isolated from IgG or IgM expressing B lymphocytes. Following transposition of LCκ libraries paired with HCγ libraries (with V_H domains from either IgG or IgM repertoires) into L11 progenitor B cells, these cellular libraries were screened for surface IgG expression and antigen-binding by simultaneous staining with an anti-human IgG-PE and hROR2-ECD-Twin-Strep detected by an anti-Strep-mAb coupled to a fluorochrome, respectively (+ hROR2-Twin-Strep). A sample lacking hROR2-ECD-Twin-Strep was stained for comparison (– hROR2-Twin-Strep). Overlays of representative plots of 10,000 cells are shown. Per library, 288 clones with high hROR2-binding relative to IgG surface levels were single-cell sorted using the indicated sort window and expanded.

Figure 4

Direct screening of supernatants from sorted clones for identification of hROR2-binding clones with high potency as an ADC. Supernatants from single-cell sorted clones were screened for hROR2 binding and additionally for potency in a secondary ADC cell killing assay. Therefore, IgG levels (dark gray bars) and binding to hROR2-ECD-Twin-Strep were measured by ELISA (lightest gray bars), presented as ratios of OD490nm hROR2-ECD-Twin-Strep-binding / IgG levels (medium gray bars). Additionally, the supernatants were used to assess potency of the antibodies for cell killing in a secondary ADC assay. For this, EMT6-hROR2 cells were incubated with a 5-fold dilution of clonal supernatant for 30 min. Next, an anti-human Fcγ coupled via a cleavable linker to PNU-159682, a potent cytotoxic small molecule, was added and incubated with the cells and clonal supernatant for 3 days. EMT6-hROR2 cells incubated with only secondary ADC, but no clonal supernatant served as a control. Viable cells were then quantified using a luminescence-based cell viability assay. Lower luminescence values [arbitrary units (a.u.)] indicate more potent killing (light gray bars). The IgG₁/Igκ library of mouse 1357 is shown as a representative. Examples of clones with strong hROR2 binding and potent (solid cell border) as well as poor in vitro killing (dashed lines) are indicated.

Figure 5

Alignment of amino acid sequences of identified hROR2-specific antibodies. Heavy chain and Kappa light chain variable regions were PCR amplified from single-cell L11 clones by reverse transcription and subjected to Sanger sequencing. The derived amino acid sequences of recovered V_H (A) and V_κ (B) are shown. Antibodies were annotated using IgBLAST. FR, framework region; CDR, complementarity-determining region. Dashes indicate gaps due to the alignment.

Figure 6

Characterization of novel hROR2-specific antibodies. (A) Identity to the closest human and mouse germline V gene sequence for heavy (HC) and light chain (LC) as a measure of “humanness” was evaluated in silico using IgBLAST. (B) Isoaffinity plot showing association (k_a) and dissociation constants (k_d) as determined by surface plasmon resonance (SPR) using clonal supernatants. Diagonal lines represent equal affinities. (C) Purified antibodies were assessed for binding to soluble human, cynomolgus monkey and mouse ROR2-ECD-Twin-Strep by ELISA.

Figure 7

Validation of ADCs. (A) hROR2 surface expression in cell lines used in this study as determined by FACS using the hROR2-specific antibody XBR2-401 (37) detected using a PE-labeled anti-human Fcγ. Black histograms, ROR2 staining; gray histograms, control staining with only the secondary antibody. (B) Following recovery of V_H and V_L sequences by RT-PCR cloning from hROR2-ECD-Twin-Strep reactive Transpo-mAb cell clones and expression in HEK293T cells, purified recombinant antibodies were site-specifically conjugated to a derivative of the potent cytotoxic payload PNU-159682 by sortase-mediated antibody conjugation (SMAC-technology^TM) to the C-termini of the heavy and light chain constant domains. These ADCs were tested for their cell killing activity on hROR2 negative (L363), and hROR2-high (T47D-hROR2) cell lines. Viability of the cells is plotted in arbitrary units (a.u.) of luminescence on the y-axis as a function of the concentration of the ADCs on the x-axis as a result of a CellTiter-Glo cell-killing assay. hROR2-negative L363 cells were used as negative controls (with an isotype-control mAb in light gray that binds to a TAA on L363). G3 and G5 designate the length of a glycine linker used in the ADCs, which has previously been shown not to affect ADC potency. Datapoints represent mean of two replicates and error bars represent SD.