Rapid, site-specific labeling of "off-the-shelf" and native serum autoantibodies with T cell-redirecting domains

Abstract

Extensive antibody engineering and cloning is typically required to generate new bispecific antibodies. Made-to-order genes, advanced expression systems, and high-efficiency cloning can simplify and accelerate this process, but it still can take months before a functional product is realized. We developed a simple method to site-specifically and covalently attach a T cell-redirecting domain to any off-the-shelf, human immunoglobulin G (IgG) or native IgG isolated from serum. No antibody engineering, cloning, or knowledge of the antibody sequence is required. Bispecific antibodies are generated in just hours. By labeling antibodies isolated from tumor-bearing mice, including two syngeneic models, we generated T cell-redirecting autoantibodies (TRAAbs) that act as an effective therapeutic. TRAAbs preferentially bind tumor tissue over healthy tissue, indicating a previously unexplored therapeutic window. The use of autoantibodies to direct the tumor targeting of bispecific antibodies represents a new paradigm in personalized medicine that eliminates the need to identify tumor biomarkers.

Fig. 1.. BiAb production mediated by a pAbBD results in six different, pure constructs that bind their respective tumor targets as expected.

(A) An anti-CD3 scFv was fused with a pAbBD. Two hours of irradiation with nondamaging long-wavelength ultraviolet (UV) light induces covalent attachment of the fusion protein to the IgG Fc region. (B) Six human antibodies—rituximab, cetuximab, trastuzumab, IgG2, IgG3, and IgG4—and the resulting BiAbs produced by photocrosslinking with pAbBD–anti-CD3 fusion protein were analyzed on a reducing SDS–polyacrylamide gel electrophoresis (SDS-PAGE). Unbound, excess pAbBD-scFv was removed via filtration. The bands represent IgG heavy chains (HC) before and after photocrosslinking. (C to E) HER2⁺ T617, epidermal growth factor receptor–positive (EGFR⁺) MDA-MB-468, and CD20⁺ HT1080 cell lines were seeded, fixed, and blocked with normal goat serum. Binding curves of photocrosslinked (C) anti-HER2 (trastuzumab) × anti-CD3, (D) anti-EGFR (cetuximab) × anti-CD3, and (E) anti-CD20 (rituximab) × anti-CD3 BiAbs and the respective monospecific antibodies from which they were derived were obtained after incubation with a fluorescent secondary antibody. Fluorescence intensity was measured using a plate reader. The dissociation constant (K_d) values were (A) 0.9 nM for the BiAb and 1.16 nM for monoclonal antibody, (B) 0.60 nM for the BiAb and 0.49 nM for the monoclonal antibody, and (C) 8.2 nM for the BiAb and 12 nM for the monoclonal antibody. All coefficient of determination (R²) values are greater than 0.9. (F) Human T cells were incubated with serial dilutions of anti-EGFR × anti-CD3 scFv BiAb, free anti-CD3 scFv, and positive control OKT3, and binding was measured via flow cytometry. The K_d values were found to be 1.89, 35, and 34 nM for OKT3, CD3 scFv, and BiAb with R² values of 0.99, 0.985, and 0.99, respectively. AU, arbitrary units.

Fig. 2.. BiAb functionality is confirmed by the T cell–mediated in vitro cytolysis targeted against HER2, EGFR, and CD20.

(A to C) T cell–mediated cytolysis of HER2⁺ T617, EGFR⁺ MDA-MB-468, and CD20⁺ HT1080 cell lines was monitored for 72 hours as a function of BiAb dose using an xCELLigence real-time cell analysis (RTCA) instrument. Increasing concentrations of anti-HER2 (trastuzumab) × anti-CD3, anti-EGFR (cetuximab) × anti-CD3, and anti-CD20 (rituximab) × anti-CD3 BiAbs were analyzed (circles), as well as mixtures of the respective unconjugated antibodies and anti-CD3 scFv (triangles), and nontargeted BiAbs (squares). The nontargeted BiAbs in (A) and (C) consisted of an anti-EGFR × anti-CD3 BiAb and were tested on EGFR-negative cell lines (HER2⁺/EGFR⁻ and CD20⁺/EGFR⁻, respectively), while in (B), an anti-Her2 × anti-CD3 BiAb was tested on an EGFR⁺/HER2⁻ cell line. Here, BiAbs were prepared using the pAbBD–anti-CD3 scFv. Equivalent studies using the nanobody format are presented in fig. S4. All assays were performed with human T cells at a 10:1 E:T ratio. BiAbs EC₅₀ values were (A) 0.038 nM, (B) 0.05 nM, and (C) 0.007 nM. All R² values were greater than 0.9. (D and E) Kinetics of T cell–mediated cytolysis of EGFR⁺ tumor cells for (D) increasing concentrations of anti-EGFR (cetuximab) × anti-CD3 BiAb or (E) a mixture of anti-EGFR cetuximab and anti-CD3 scFv. All assays were performed with human T cells using an E:T of 10:1. (F) T cell–mediated cytolysis of EGFR⁺ cells with increasing E:T ratios, 12 hours after treatment with 0.1 nM EGFR-targeted BiAbs produced with pAbBD–anti-CD3 scFv or 0.1 nM cetuximab mixed with 0.2 nM free anti-CD3 scFv.

Fig. 3.. pAbBD-mediated production of TRAAbs creates personalized, functional targeted therapeutics without needing to identify tumor markers.

(A) Endogenous extracted from the serum of mice vaccinated with Nalm-6 B cells were converted into TRAAbs via site-specific, covalent photocrosslinking of the pAbBD–anti-CD3 fusion protein. The tumor killing capability of the resulting BiAbs was evaluated in vitro and in vivo using immunodeficient mice engrafted with human T cells. (B) Whole-tumor vaccination of C57BL/6 immunocompetent mice with CD19⁺ Nalm-6 B cell leukemia was completed as follows: 2 × 10⁶ cells were intraperitoneally (i.p.) injected twice a week for 2 weeks, followed by 2 weeks off. This 28-day cycle was repeated twice before collecting the entire blood volume via cardiac puncture. Blood samples were also collected after the second and fourth weeks. (C) Blood samples of vaccinated mice were centrifuged to extract serum, and all IgGs, including tumor-specific autoantibodies (AAbs), were purified using protein G agarose resin. TRAAbs were produced via photocrosslinking with a pAbBD–anti-CD3 nanobody. Heavy chain (HC) and light chain (LC) are shown before and after photocrosslinking on a reducing SDS-PAGE. (D) Luciferase-expressing CD19⁺ Nalm-6 B cell leukemia cells were incubated with human T cells, using a 5:1 E:T, and cytolysis was analyzed using the Steady-Glo Luciferase Assay System (Promega) 24 hours after antibody treatment. Treatments included BiAbs that were generated by photocrosslinking pAbBD–anti-CD3 nanobodies to (i) AAbs extracted from vaccinated C57BL/6 mice (i.e., TRAAbs), (ii) IgGs extracted from WT C57BL/6 mice, and (iii) control anti-CD19 IgGs (MAB1794, EMD Millipore). A T cell only control was also tested. The EC₅₀ values were found to be 0.92 nM for TRAAbs, using AAbs that were collected 8 weeks after the first Nalm-6 injection (R² = 0.99), and 0.90 nM for the anti-CD19 × anti-CD3–positive control (R² = 0.95).

Fig. 4.. TRAAb therapeutic efficacy is confirmed by in vivo tumor suppression and improved survival.

(A to D) Immunodeficient NSG mice were injected with 0.5 M Nalm-6 B cell leukemia cells and (A) left untreated, or treated with 10 M human T cells and a total dose of 0.5 mg/kg of (B) BiAbs produced from IgGs extracted from WT nonvaccinated mice, (C) mixed AAbs and anti-CD3 nanobody, or (D) TRAAbs. Treatment was evenly distributed along days 1, 3, and 5, as was 1 mg of background, WT polyclonocal murine IgG (BE0093, Bio X Cell). Tumor growth was tracked twice per week via luciferase expression and d-luciferin (PerkinElmer) injections using the IVIS Ilumina system. (C and D) p/s photons/s. (E) Averaged bioluminescence readings for groups depicted in (A) to (D). n = 6 or 7. At day 28, *P < 0.05 or smaller between TRAAb and all controls. (F) Sixty-day survival Kaplan-Meier curves.

Fig. 5.. Functionality of TRAAbs produced using syngeneic, endogenous antibodies.

(A) Endogenous antibodies were extracted from the sera of BALB/c mice growing syngeneic CT26 colorectal tumors subcutaneously, as well as WT BALB/c mice. The purified IgG were photocrosslinked with pAbBD–anti-CD3, to create TRAAbs, and run on a denaturing and native SDS-PAGE. (B) Endogenous IgGs from mice with CT26 tumors (AAbs) bind CT26 cells specifically, while no significant binding was observed with control WT IgGs from healthy BALB/c mice and nontargeted anti-human HER2 (Herceptin). (C) TRAAbs produced using IgGs from CT26 tumor–bearing mice yielded dose-dependent tumor cytolysis 24 hours after treatment, as does a positive control targeted against mouse EGFR. A negative control composed of T cell recruiting WT IgGs led to minimal cytolysis and only at the highest concentration tested. All syngeneic in vitro studies were completed using an E:T ratio of 8:1.

Fig. 6.. Circulating endogenous antibodies preferentially target tumor tissue over healthy tissues in two syngeneic models.

(A) Immunofluorescent staining of syngeneic colorectal CT26 tumor tissue and healthy tissue of major organs where endogenous antibodies isolated from CT26 tumor–bearing mice were used as the primary antibody. The fluorescent signal was quantified and normalized to controls using isotype control secondary antibodies, n = 5, P < 0.001 or smaller. (B) Replicate of (A) using positive control cell line CT26-HER2 that was engineered to express human HER2/neu receptor. Here, staining was done using murine anti-human HER2/neu as a primary antibody. n = 3, P < 0.01. (C) replicates of (A) in triple-negative breast cancer 4T1 model. n = 5, P < 0.01. (D and E) Representative images used for above quantifications of CT26 and 4T1 circulating antibodies bound on tumor tissues versus healthy tissues of major organs. Scale bar represents 500 microns.