We have developed MGD007 (anti-glycoprotein A33 x anti-CD3), a DART protein designed to redirect T cells to target gpA33 expressing colon cancer. The gpA33 target was selected on the basis of an antibody-based screen to identify cancer antigens universally expressed in both primary and metastatic colorectal cancer specimens, including putative cancer stem cell populations. MGD007 displays the anticipated-bispecific binding properties and mediates potent lysis of gpA33-positive cancer cell lines, including models of colorectal cancer stem cells, through recruitment of T cells. Xenograft studies showed tumor growth inhibition at doses as low as 4 μg/kg. Both CD8 and CD4 T cells mediated lysis of gpA33-expressing tumor cells, with activity accompanied by increases in granzyme and perforin. Notably, suppressive T-cell populations could also be leveraged to mediate lysis of gpA33-expressing tumor cells. Concomitant with CTL activity, both T-cell activation and expansion are observed in a gpA33-dependent manner. No cytokine activation was observed with human PBMC alone, consistent with the absence of gpA33 expression on peripheral blood cell populations. Following prolonged exposure to MGD007 and gpA33 positive tumor cells, T cells express PD-1 and LAG-3 and acquire a memory phenotype but retain ability to support potent cell killing. In cynomolgus monkeys, 4 weekly doses of 100 μg/kg were well tolerated, with prolonged PK consistent with that of an Fc-containing molecule. Taken together, MGD007 displays potent activity against colorectal cancer cells consistent with a mechanism of action endowed in its design and support further investigation of MGD007 as a potential novel therapeutic treatment for colorectal cancer. Mol Cancer Ther; 17(8); 1761–72. ©2018 AACR.

Colorectal cancer is the second leading cause of death due to cancer in the United States with 49,190 deaths expected in 2016 and 134,490 expected new cases. Approximately 50% to 60% of patients with colorectal cancer have metastatic disease at diagnosis with common sites of involvement, including liver, lymph nodes, lung, peritoneum, and soft tissues. Despite some success with existing agents and combination regimens, including those targeting angiogenesis and EGFR, the prognosis of patients with metastatic colorectal cancer (mCRC) remains extremely poor overall (1).

Recent reports however have suggested that newly diagnosed colorectal cancer patients with evidence of immune infiltration in their tumors may have a more favorable clinical prognosis (2–4). Collectively, these observations have led to the generation of a set of criteria, known as the Immunoscore (5), that is now being evaluated as both a prognostic and predictive biomarker in patients with colorectal carcinoma. Furthermore, programmed death-1 (PD-1) blockade with pembrolizumab has demonstrated clinical efficacy in the subset of patients with colorectal cancer that are mismatch repair deficient (6). However, the majority of patients with colorectal cancer (>85%) are mismatch repair proficient and do not respond to immune checkpoint blockade, indicating the need for alternate strategies to leverage T-cell–mediated responses for antitumor activity. One such approach being explored across multiple cancer types are bispecific antibodies designed to recruit host T cells and harness their cytolytic activity to selectively eradicate the target cancer cell population (7). Indeed, Blinatumomab, an antibody-based bispecific T-cell engager (BiTE)–directed against CD3 and CD19, is an approved immunotherapeutic strategy capable of enabling the patient's own T lymphocytes to eliminate leukemia cells (8). However, the need for continuous intravenous infusion due to its short half-life represents significant limitations of blinatumomab clinical use.

To address the functional and structural limitations of existing bispecific molecules, we have developed the DART platform that displays favorable structural and biological properties, including stability and optimal redirect T-cell killing of malignant tumor cells (9–11). Previous studies have also demonstrated successful application of DART molecules for the targeting of B cells through either recruitment of natural killer cells (12) or by co-ligation of inhibitory and activating receptor pathways (13). Importantly, DART molecules can also be tailored to incorporate an Fc domain to enhance half-life and support convenient intermittent dosing rather than continuous infusion (14, 15). In the present study, we have explored the potential for DART molecules to recruit the cytolytic activity of human T cells against colorectal cancer cells.

Dissection of colorectal cancer progression has revealed compelling evidence for the critical role played by the cancer stem cell (CSC), a self-renewing, immortal cell that maintains cancer by supporting tumor growth and differentiation (16, 17). Although these observations have fueled interest in targeting eradication of the colorectal CSC (18–20), this is countered by the apparent plasticity of human cancer in the face of changing tumor niche to revert from non-CSC to CSC (21, 22) calling into question strategies that solely target the putative CSC population. With this in mind we set out to identify a colorectal cancer cell surface target expressed on both the CSC and differentiated tumor cell population. Characterization of mAbs generated from murine immunization of a colorectal cancer–derived cancer stem-like cell (CSLC) line identified glycoprotein A33 (gpA33), a cell surface antigen previously subjected to therapeutic mAb targeting (23, 24) as being expressed across a panel of CSLCs derived from a various stages of colorectal cancer, in addition to exhibiting 100% penetrance across >50 primary and metastatic colorectal cancer specimens. To enable therapeutic targeting of gpA33, we generated MGD007, gpA33 x CD3 DART protein designed to co-engage gpA33-expressing colorectal cancer cells with CD3-expressing T cells and herein characterized its biological activity in vitro and in vivo.

Cancer stem-like cell line generation and characterization

All human specimens were obtained from Kaiser Permanente (KSPC #4548), the National Disease Research Interchange (NDRI) or the Cooperative Human Tissue Network (CHTN) with institutional review board approval and appropriate written informed consent for tissue acquisition and use. Colorectal cancer tissue was shipped on ice and received within 24 hours of excision. Tissue with visible microbial contamination on receipt was discarded. Tumors were minced into small (<1mm) fragments and treated with collagenase/dispase (Roche Applied Science) until small aggregates of dissociated cells were observed; dispersing all tissue to single cells severely decreased viability. The conditions appropriate for tissue stem/progenitor cells were previously optimized using a stepwise approach (25, 26); these conditions were further modified empirically for the optimal isolation and growth of colon cancer cells. Viable dissociated cells were plated on tissue culture plates pre-coated with fibronectin and laminin in F12/DME (50:50 v/v) medium supplemented with: recombinant human insulin (10 μg/mL), transferrin (10 μg/mL), EGF (5 ng/mL), ethanolamine (10−9 mol/L), phosphoethanolamine (10−9 mol/L), tri-iodothyronine (T3, 10−12 mol/L), selenium (2.5 × 10−8 mol/L), hydrocortisone (2.5 × 10−8 mol/L), vitamin E (5 μg/mL), glucagon (50 ng/mL), and gastrin-1 (100 ng/mL). Cell line authentication was determined by sixteen-locus short tandem-repeat (STR) analysis performed on the original tumor specimen and longitudinally during cell line development using the AmpFℓSTR Identifiler polymerase chain reaction (PCR) Amplification Kit (Applied Biosystems; Supplementary Table S1). Mutation analysis of KRAS exon 2, BRAF exon 15, and PIK3CA exons 9 and 23, and the Mutation Cluster Region (MCR) of APC exon 15 was performed by sequencing amplified genomic DNA (27, 28). For flow cytometry analyses, CSLC were harvested with collagenase/dispase or trypsin/EDTA, washed, resuspended in HBSS (Invitrogen) with 1% BSA (Rockland Immunochemicals) and stained with indicated antibodies. To enable in vitro cell differentiation, CSLCs were plated on the basis of conditions previously reported for the differentiation of LGR5+ (29) or EphB2-sorted (30) murine crypt cells. Briefly, small aggregates or sorted single cells were mixed with Matrigel overlaid with F12/DME supplemented with B27 and N2 media supplements (Invitrogen). Organoids were prepared for frozen tissue immunohistochemistry and stained for carbonic anhydrase II (CAII, enterocytes); chromogranin A (enteroendocrine cells); and MUC2 (goblet cells). To evaluate tumorigenicity, CSLC lines were implanted under sub-renal capsule of immune deficient mice. Initial patient sample from which CSLC was derived and excised tumor xenograft tissue were fixed in 10% neutral formalin, embedded in paraffin blocks and sectioned for H&E staining.

Identification of anti-gpA33 mAb RECA47

The mouse RECA47mAb was derived from an intact whole-cell immunization of the RECA020108 CSLC performed as previously described (31). Briefly, Balb/c mice received a primary footpad immunization with 50 μL of RECA020108 (2 x 106 cell/mL) in RIBI adjuvant followed by a booster dose one week before lymph node collection and fusion with LB653 myeloma cells. Hybridoma supernatants demonstrating reproducible binding to RECA020108 by flow cytometry were selected for purification and additional flow cytometry and IHC analyses, leading to identification of RECA47mAb. To identify the binding antigen for RECA47mAb, a mammalian cDNA expression library of RECA020108 CSLC was constructed, a battery of DNA pools prepared and expressed in CHO cells, and screened by immunohistochemistry with RECA47mAb by limited dilution cloning. A positive clone was identified and DNA sequencing revealed identity to gpA33. Humanized RECA47 VH and VL sequences are based on the CDRs from mouse RECA47mAb and human frameworks (FR) derived from human germline.

Characterization of MGD007 binding properties

Binding of MGD007 to soluble human and cynomolgus monkey CD3 and gpA33 receptors was evaluated by surface plasmon resonance (SPR): MGD007 binding to immobilized CD3 protein was analyzed (in duplicates) at concentrations of 0, 6.25, 12.5, 25, 50, and 100 nmol/L. Binding to gpA33 was evaluated using human (12.5 nmol/L) gpA33 His-tagged proteins expressed and purified from Chinese hamster ovary (CHO) cells. For flow cytometry analyses, MGD007 or control DART with 0.5 to 1 x 106 cells/mL (100 μL) of cancer cells, engineered CHO cells, or human or cynomolgus T cells were analyzed by flow cytometry with biotin-conjugated anti-EK antibody mixed with APC-streptavidin.

Functional assays

Redirected killing of gpA33-expressing cell lines using human PBMC (E:T = 30:1) or T-cell subsets (E:T ≤ 10:1) was evaluated as previously described using either LDH readout or cell lines engineered to constitutively express luciferase (32, 33). Colo205, SW948, and LS174T cancer cell lines were obtained from the ATCC; JIMT-1 was obtained from DSMZ (Braunsschweig, Germany); RECA0201-GF CSLC line, stably transduced to constitutively express luciferase and GFP was generated at MacroGenics. Cell lines were authenticated by STR analyses, determined to be mycoplasma free, and passaged for less than 3 months. T-cell activation, intracellular granzyme B and perforin levels and T-cell proliferation were determined as described previously (32). Human T cells, CD4+ T cells and CD8+ T cells were isolated via negative selection from PBMCs using Untouched human T-cell isolation, CD4+ T-cell isolation, or CD8+ T-cell isolation kits (Life Technologies). Suppressive T cells were isolated using human regulatory T-cell enrichment kit (Stem cell technologies) and expanded ex vivo as previously described (34). For indicated experiments a previously described (32) control DART protein (single arm CD3) comprising an anti-fluorescein mAb 4-4-20 specificity rather than anti-gpA33 was employed. Non-linear regression analyses were used to fit curves in GraphPad Prism6. Flow cytometry data were performed using Flowjo v9.3.3 software (Treestar, Inc.).

Co-mix in vivo models

All studies were reviewed and approved by MacroGenics' Institutional Animal Care and Use Committee (IACUC). Female NOD/SCID mice were used for this study (n = 8/group). Human T cells were isolated from heparinized whole blood using RosetteSep T-cell isolation kit (Stemcell technologies). The purified T cells were subsequently activated by exposing the cells to anti-CD3 (OKT-3; 1 μg/mL) and anti-CD28 (66 μg/mL) antibodies for a period of 48 hours and maintained in presence of IL-2 (7.6 ng/mL) for up to 3 weeks. The human T cells and tumor cells were combined at a ratio of 1:1 (5 x 106 cells each) and suspended in 200 μL of sterile saline and injected subcutaneously on day 0. The vehicle control (0.5% sterile saline containing 0.5% BSA) or MGD007 was administered intravenously via tail vein injections (100 μL) on Days 0, 1, 2, and 3. Individual tumor dimensions (length × width) were measured using calipers daily from Day 7 to 24 and calculated as described previously (10, 32).

Monkey study/PK assay

Study was performed at Charles River Laboratories (Reno, NV) under IACUC guidelines. Pharmacokinetics was evaluated in cynomolgus monkeys (n = 4/sex/group) after intravenous administration of 30 or 100 μg/kg MGD007 over 2 hours once weekly for 4 weeks. All animals also received vehicle control for the 1st infusion on Day 1. A two-compartment model with constant intravenous infusion was used for PK analysis.

Identification of gpA33 as a cell surface antigen universally expressed on putative colorectal cancer stem cell populations

To enable identification of cell surface antigens expressed on the putative colorectal cancer stem cell population, novel cell lines were developed from freshly resected human colorectal cancer samples using conditions designed to enrich and maintain putative stem cell populations (detailed in Materials and Methods). A panel of 9 CSLC lines were derived, representative of various stages of colorectal cancer differentiation, including both KRAS WT and mutated cancer, two lines derived from metastatic lesions and one from a high microsatellite instable (MSI-H) specimen (Supplementary Table S2). Under serum-free, growth factor-supplemented defined conditions, the CSLC can be maintained indefinitely as semi-adherent cultures on a fibronectin and laminin matrix, while under conditions that promote colon crypt differentiation, the CSLC have capacity to differentiate into organoids comprising the principal cell types observed in the colon and differentiated colorectal tumors (Fig. 1A; Supplementary Fig. S1). When implanted in immune deficient mice under the sub renal capsule, the RECA CSLC form tumors that fully recapitulate the morphologic and phenotypic characteristics of the patients' original tumors (Fig. 1A). Taken together, the CSLC lines therefore exhibit the key properties associated with the cancer stem cell: self-renewal, differentiation and tumor initiation. To identify antibodies that recognize cell surface antigens expressed on the CSLC, a whole-cell murine immunization was performed with the RECA020108 CSLC. Hybridoma supernatants demonstrating cell surface binding to the immunizing CSLC were purified and analyzed by flow cytometry across the broader panel of CSLC lines and by IHC to determine their reactivity toward CSLC xenograft, primary and metastatic colorectal cancer tissues. One said mAb, RECA47mAb, displayed such a profile indicative of reactivity to a cell surface antigen universally expressed in colorectal cancer (Fig. 1B). Expression cloning identified the antigen for this antibody as glycoprotein A33 (gpA33), which was confirmed by immunoprecipitation and western blotting, with SPR analyses revealing high-affinity binding to recombinant gpA33 (Fig. 1C). The expression of gpA33 across the CSLC panel, regardless of their mutational status or histological derivation is distinguished from CD133 and CD44, generally regarded as “canonical” CSC markers, which are not expressed on a subset of the CSLC (Fig. 1D). Furthermore, IHC analyses of a panel of approximately 50 individual colorectal cancer tumor specimens (both primary and metastatic, Fig. 1E; Supplementary Table S3) revealed all were reactive at a 2–3+ level to RECA47mAb whereas IHC across a panel of normal adult human tissues confirmed reactivity restricted to intestinal epithelium (Supplementary Fig. S2), a collective binding profile consistent with that of a gpA33 reactive antibody (35, 36). On the basis of its universal expression profile across models of colorectal cancer stem cells together with homogenous expression within tumor specimens, gpA33 was selected for a DART-based T-cell immunotherapy for the treatment of metastatic colorectal cancer.

Figure 1.

Colorectal cancer stem-like cell (CSLC) panel and identification of gpA33 as universally expressed colorectal cancer cell surface target. A, Morphological image of RECA020108, representative of nine independent colorectal cancer stem-like cell (CSLC) lines expanded in vitro under serum-free culturing conditions from freshly isolated colorectal cancer tumor resections (top right). Single-cell cloning of RECA020108 in Matrigel supplemented with growth factors, yields organoid structures (bottom right) whereas implant under the subrenal capsule of immune deficient mice yielded differentiated xenografts (bottom left, H&E) indistinguishable from the original patient tumor sample from which the line was derived (top left, H&E). B, Whole-cell immunizations in Balb/c mice with the CSLC line RECA020108 identified mAb RECA47 displaying cell surface reactivity to RECA020108 by flow cytometry (left) and by IHC to RECA020108-derived xenografts (right). C, Western blot analyses (left) performed with a previously characterized gpA33 antibody directly on COLO205 lysate (1), cell extract immunoprecipitated with control gpA33 antibody (2) or RECA47mAb (3) or a negative control mAb (4) confirmed reactivity of RECA47mAb with a protein species corresponding to the MW of gpA33. SPR analyses (right) demonstrating binding affinity of soluble gpA33 to RECA47mAb. D, IHC-binding profile of RECA47mAb (top row) and flow cytometry profile (bottom row) of CD44 and CD133 expression across four representative CSLC lines (RECA060810 (KRAS mt); RECA062409 (KRAS mt); RECA082509 (KRAS WT; MSI-H); RECA090422 (KRAS WT). E, RECA47mAb reactivity with colorectal cancer specimens by IHC, including matched pairs of primary (left) and metastatic (metastasis to liver, right) colon adenocarcinoma).

Figure 1.

Colorectal cancer stem-like cell (CSLC) panel and identification of gpA33 as universally expressed colorectal cancer cell surface target. A, Morphological image of RECA020108, representative of nine independent colorectal cancer stem-like cell (CSLC) lines expanded in vitro under serum-free culturing conditions from freshly isolated colorectal cancer tumor resections (top right). Single-cell cloning of RECA020108 in Matrigel supplemented with growth factors, yields organoid structures (bottom right) whereas implant under the subrenal capsule of immune deficient mice yielded differentiated xenografts (bottom left, H&E) indistinguishable from the original patient tumor sample from which the line was derived (top left, H&E). B, Whole-cell immunizations in Balb/c mice with the CSLC line RECA020108 identified mAb RECA47 displaying cell surface reactivity to RECA020108 by flow cytometry (left) and by IHC to RECA020108-derived xenografts (right). C, Western blot analyses (left) performed with a previously characterized gpA33 antibody directly on COLO205 lysate (1), cell extract immunoprecipitated with control gpA33 antibody (2) or RECA47mAb (3) or a negative control mAb (4) confirmed reactivity of RECA47mAb with a protein species corresponding to the MW of gpA33. SPR analyses (right) demonstrating binding affinity of soluble gpA33 to RECA47mAb. D, IHC-binding profile of RECA47mAb (top row) and flow cytometry profile (bottom row) of CD44 and CD133 expression across four representative CSLC lines (RECA060810 (KRAS mt); RECA062409 (KRAS mt); RECA082509 (KRAS WT; MSI-H); RECA090422 (KRAS WT). E, RECA47mAb reactivity with colorectal cancer specimens by IHC, including matched pairs of primary (left) and metastatic (metastasis to liver, right) colon adenocarcinoma).

Close modal

gpA33 x CD3 DART (MGD007) design and biophysical properties

MGD007, a gpA33 x CD3-bispecific antibody–based molecule, was constructed using the DART platform, incorporating monovalent binding for both gpA33 and CD3 in addition to an IgG1 Fc domain to provide antibody-like pharmacokinetics. It is heterotrimeric—composed of three chains co-expressed and assembled through disulfide bonds and non-covalent forces (Fig. 2A). Chains 1 and 2 provide the dual-antigen-binding portion of MGD007 with the anti-CD3 Fv region derived from humanized XR32 (10) and the anti-gpA33 specificity based on humanized RECA47mAb. Chains 1 and 3 provide the Fc chains. Chains 1 and 2 form a heterodimer through a disulfide bond at the C-termini of the chains and by virtue of oppositely charged coiled–coil sequences (E-coil and K-coil). Chains 1 and 3 are covalently linked by two disulfide bonds in the Fc hinge region. To prevent homodimerization of Chains 1 and 3, the knob (T366W) and hole (T366S/L368A/Y407V) mutations have been incorporated in the respective CH3 region of each Fc. To facilitate the removal of any remaining homodimers of Chain 3 during purification, the H435R mutation has been incorporated in the Fc CH3 region. The Fc CH2 regions in Chains 2 and 3 contain Ala, Ala substitutions (L234A/L235A) to markedly reduce or eliminate FcγR and complement binding, while maintaining neonatal FcR (FcRn) binding to take advantage of the IgG salvage pathway mediated by this receptor. MGD007 was expressed in CHO cells and purified using Protein A–based chromatography with purity and structural integrity demonstrated by reduced capillary electrophoresis and SE-HPLC (Fig. 2B). The reduced capillary electrophoresis analysis confirmed that MGD007 is primarily composed (99%) of three protein chains and the SE-HPLC analysis demonstrated the correct apparent molecular weight (approximately 110 kDa). Concentration-dependent binding of MGD007 to CHO cells transfected with either human or cynomolgus monkey gpA33 was confirmed, while no MGD007 binding was observed on parental CHO cells (Fig. 2C). Likewise, MGD007 showed similar binding to both human and cynomolgus monkey T cells (Fig. 2D). SPR analysis using recombinant soluble human antigens revealed MGD007 binds with KD values of 2.2 nmol/L to gpA33 and 23 nmol/L to CD3 (Supplementary Table S4).

Figure 2.

MGD007 molecular structure and bispecific binding to gpA33 and CD3. A, MGD007 is composed of three chains covalently linked by disulfide bonds (50). Chains 1 and 2 form a heterodimer of oppositely charged coiled–coiled sequences stabilized through a C-terminal disulfide bond. Chains 1 and 3 are linked by two disulfide bonds in the Fc hinge region. To prevent homodimerization of Chains 1 and 3, knob-into-hole mutations have been incorporated in the CH3 region of the Fc. Ala, Ala mutations were also introduced into the Fc region to limit unwanted FcγR interactions, but retain FcRn binding to enhance circulating half-life. B, MGD007 purity and structural integrity is demonstrated by reduced capillary electrophoresis and SE-HPLC. The reduced capillary electrophoresis analysis confirms the presence of the three protein chains whereas the SE-HPLC analysis demonstrates the correct apparent molecular weight. C, Flow cytometry analyses of MGD007 binding to CHO cells transfected with human or cynomolgus monkey gpA33. D, Flow-cytometry analyses of MGD007 binding to human T cells and cynomolgus monkey T cells. Gray: unstained cells; blue: secondary detection reagents alone; red: MGD007 binding.

Figure 2.

MGD007 molecular structure and bispecific binding to gpA33 and CD3. A, MGD007 is composed of three chains covalently linked by disulfide bonds (50). Chains 1 and 2 form a heterodimer of oppositely charged coiled–coiled sequences stabilized through a C-terminal disulfide bond. Chains 1 and 3 are linked by two disulfide bonds in the Fc hinge region. To prevent homodimerization of Chains 1 and 3, knob-into-hole mutations have been incorporated in the CH3 region of the Fc. Ala, Ala mutations were also introduced into the Fc region to limit unwanted FcγR interactions, but retain FcRn binding to enhance circulating half-life. B, MGD007 purity and structural integrity is demonstrated by reduced capillary electrophoresis and SE-HPLC. The reduced capillary electrophoresis analysis confirms the presence of the three protein chains whereas the SE-HPLC analysis demonstrates the correct apparent molecular weight. C, Flow cytometry analyses of MGD007 binding to CHO cells transfected with human or cynomolgus monkey gpA33. D, Flow-cytometry analyses of MGD007 binding to human T cells and cynomolgus monkey T cells. Gray: unstained cells; blue: secondary detection reagents alone; red: MGD007 binding.

Close modal

MGD007-mediated T-cell lysis of gpA33-expressing colon cancer cells and inhibition of murine xenografts

MGD007-mediated in vitro lysis of gpA33-expressing colorectal cancer cells in the presence of either human PBMC donor or freshly isolated T cells is shown in Fig. 3A–D. Although gpA33+ cell lines (LS174T, Colo205) and a representative CSLC (RECA020108-GF) line were all efficiently lysed by MGD007, no cytotoxicity was observed against a human breast cancer cell line (JIMT-1) lacking gpA33 expression (Supplementary Fig. S3) or with the single-arm, CD3-engaging control DART molecule lacking gpA33 targeting specificity. Notably, both Colo205 or the RECA020108-GF CSLC line were effectively completed lysis with MGD007 consistent with uniform expression of gpA33 (Fig. 3B-C). In addition, evaluation of multiple independent donor T cells demonstrated all were supportive of MGD007 activity against Colo205 cells with EC50 values spanning single to double digit ng/mL (Fig. 3D). Inhibition of tumor growth by MGD007 was evaluated in vivo in NOD/SCID mice implanted SC with Colo205 or LS174T human colorectal tumor cells in the presence of activated human T cells. Although the lowest MGD007 dose evaluated (0.8 μg/kg) resulted in no tumor growth inhibition in either model, treatment with MGD007 at doses ≥4 μg/kg resulted in significant inhibition of both Colo205 and LS174T tumor growth (Fig. 3E), with mice treated at ≥ 20 μg/kg showing no apparent tumor growth in either model. LS174T or Colo205 tumors in the vehicle or control DART molecule–treated groups demonstrated a monophasic growth profile for the duration of the study.

Figure 3.

MGD007 mediates redirected T-cell killing of gpA33-expressing colorectal cancer cells. MGD007-mediated cytotoxicity against (A) gpA33+ LS174T colorectal cancer cell in presence of freshly isolated human PBMC (E:T = 30:1; 24 hours) as determined by LDH release assay; (B) gpA33+ luciferase transfected Colo205-Luc colorectal cancer cell line in presence of freshly isolated human PBMC (E:T = 30:1; 24 hours); (C) gpA33+ luciferase-transduced RECA020108-GF colorectal CSLC in presence of freshly isolated human T cells (E:T = 10:1; 48 hours); and (D) gpA33+ luciferase transfected Colo205-Luc colorectal cancer cell line in presence of freshly isolated T cells obtained from four independent donors (E:T cell ratio = 10:1; 24 hours) as determined by decrease in luciferase fluorescence. No lytic activity was observed against JIMT1, a gpA33 breast cancer cell line. E, Female NOD/SCID mice (n = 8/group) were implanted SC with activated human T cells co-mixed with LS174T or Colo205 tumor cells (E:T = 1:1) on day 0 followed by treatment with vehicle control, control DART, or 0.8 to 100 μg/kg MGD007 on days 0–3 administered intravenous. Data represented as the mean ± SEM. Statistical analyses were carried out between treated and control groups comparing tumor volumes using two-way ANOVA followed by a Bonferroni post hoc (*, P < 0.001).

Figure 3.

MGD007 mediates redirected T-cell killing of gpA33-expressing colorectal cancer cells. MGD007-mediated cytotoxicity against (A) gpA33+ LS174T colorectal cancer cell in presence of freshly isolated human PBMC (E:T = 30:1; 24 hours) as determined by LDH release assay; (B) gpA33+ luciferase transfected Colo205-Luc colorectal cancer cell line in presence of freshly isolated human PBMC (E:T = 30:1; 24 hours); (C) gpA33+ luciferase-transduced RECA020108-GF colorectal CSLC in presence of freshly isolated human T cells (E:T = 10:1; 48 hours); and (D) gpA33+ luciferase transfected Colo205-Luc colorectal cancer cell line in presence of freshly isolated T cells obtained from four independent donors (E:T cell ratio = 10:1; 24 hours) as determined by decrease in luciferase fluorescence. No lytic activity was observed against JIMT1, a gpA33 breast cancer cell line. E, Female NOD/SCID mice (n = 8/group) were implanted SC with activated human T cells co-mixed with LS174T or Colo205 tumor cells (E:T = 1:1) on day 0 followed by treatment with vehicle control, control DART, or 0.8 to 100 μg/kg MGD007 on days 0–3 administered intravenous. Data represented as the mean ± SEM. Statistical analyses were carried out between treated and control groups comparing tumor volumes using two-way ANOVA followed by a Bonferroni post hoc (*, P < 0.001).

Close modal

MGD007-mediated cytotoxicity is associated with T-cell activation and can be supported by various T-cell subsets

To determine the relationship of MGD007-mediated cytotoxicity with T-cell activation and cytokine release, a parallel analysis of each functional activity using two gpA33-expressing model colorectal cancer cell lines (LS174T and SW948) was performed (Fig. 4A). Flow cytometry analyses confirmed MGD007 cell surface binding to both cells with slightly higher-level binding observed on SW948 (Supplementary Fig. S3). Concomitant with MGD007 mediated cytotoxicity (left hand), the CD69 T-cell activation marker was upregulated on CD4 and CD8 T cells in a dose-dependent manner upon exposure to gpA33-expressing cells (Fig. 4A, middle). In addition, the level of TNF-α, and IFNγ in supernatants of PMBC co-cultured with colorectal cancer cell lines also increased in presence of MGD007 (Fig. 4A, right) but not with PBMC alone, consistent with the restricted expression of gpA33 to intestinal cells (36).

Figure 4.

MGD007-mediated cytotoxicity is associated with T-cell activation and cytokine release and is supported by various CD3 T-cell sub-populations, including Tregs. A, MGD007-mediated cytotoxicity against gpA33+ SW498 and LS174T colon cancer cell lines in presence of human PBMC (E:T = 30:1; 24 hours) as determined by LDH release (left); concomitant T-cell activation as measured by the percentage of CD8 and CD4 cells detected as CD69+ (middle) and increased IFN-γ and TNF-α in supernatants from samples treated with 10 μg/mL MGD007 (right). B, MGD007-mediated cytotoxicity against gpA33+ luciferase transfected Colo205-Luc colorectal cancer cell line in presence of purified human T cells from same donor (CD3, CD8, or CD4 as indicated; E:T = 10:1, 48 hours) as determined by decrease in luciferase fluorescence (left graph). Dose-dependent upregulation of granzyme B (GB) and perforin levels (MFI plotted net of background levels observed with control DART) in both CD8 and CD4 human T-cell subsets (right graph) following incubation of MGD007 with Colo205 and purified T cells (E:T = 10:1; 24 hours). C, MGD007-mediated cytotoxicity against luciferase transfected Colo205-Luc colorectal cancer cell line in presence of either conventional CD3 T cells or suppressive CD4+/FOXP3+ T cells (E:T = 10:1; 24 hours). No cytotoxicity was mediated by control DART (single arm-CD3).

Figure 4.

MGD007-mediated cytotoxicity is associated with T-cell activation and cytokine release and is supported by various CD3 T-cell sub-populations, including Tregs. A, MGD007-mediated cytotoxicity against gpA33+ SW498 and LS174T colon cancer cell lines in presence of human PBMC (E:T = 30:1; 24 hours) as determined by LDH release (left); concomitant T-cell activation as measured by the percentage of CD8 and CD4 cells detected as CD69+ (middle) and increased IFN-γ and TNF-α in supernatants from samples treated with 10 μg/mL MGD007 (right). B, MGD007-mediated cytotoxicity against gpA33+ luciferase transfected Colo205-Luc colorectal cancer cell line in presence of purified human T cells from same donor (CD3, CD8, or CD4 as indicated; E:T = 10:1, 48 hours) as determined by decrease in luciferase fluorescence (left graph). Dose-dependent upregulation of granzyme B (GB) and perforin levels (MFI plotted net of background levels observed with control DART) in both CD8 and CD4 human T-cell subsets (right graph) following incubation of MGD007 with Colo205 and purified T cells (E:T = 10:1; 24 hours). C, MGD007-mediated cytotoxicity against luciferase transfected Colo205-Luc colorectal cancer cell line in presence of either conventional CD3 T cells or suppressive CD4+/FOXP3+ T cells (E:T = 10:1; 24 hours). No cytotoxicity was mediated by control DART (single arm-CD3).

Close modal

To evaluate the contribution of T-cell subsets to MGD007-mediated target cell killing, T cells from the same donor were purified and CTL assays were performed in parallel with total T cells (CD3+) as well as CD4+ or CD8+ subpopulations as effector cells and Colo205 as target cells. As shown in Fig. 4B, all three T-cell populations supported lysis of luciferase-expressing Colo205 target cells with potency in the relative order of CD8 > CD3 > CD4. The enhanced potency with CD8 T cells was mirrored with increased dose-dependent upregulation of granzyme B and perforin levels in human CD8+ T cells compared with CD4+ T cells (Fig. 4C). Evaluation of the lytic activity of an in vitro expanded population of CD4+Foxp3+ Tregs, with confirmed suppressive activity against conventional T cells (Supplementary Fig. S4), revealed they can also be leveraged by MGD007 to lyse gpA33-expressing target cells albeit with an approximate 10-fold potency reduction (Fig. 4D) compared with unfractionated T cells. In contrast, control DART (single arm-CD3) mediated no cytotoxicity with either conventional CD3 or suppressive T cells.

Prolonged exposure to MGD007 drives expansion of T cells with memory phenotype that retain cytolytic activity but reduced cytokine response

T-cell expansion is a hallmark response to T-cell activation supported by CD3 bispecifics following co-engagement of CD3 with tumor-specific antigen; consistent with this anticipated response T cells underwent replication in the presence of MGD007 and gpA33 target cells as determined following 72- and 96-hour incubation (Fig. 5A). In contrast, no proliferation of T cells was observed in the presence of the control DART molecule. During this timeframe, expression of cell surface markers associated with both T-cell activation and checkpoint inhibition (CD25, PD-1, LAG-3) were also upregulated (Fig. 5B), whereas evaluation of the T-cell phenotype following prolonged incubation over 7 days demonstrated a drive away from naïve T-cell phenotype to central and effector memory (TCM, TEM) phenotypes (Fig. 5C). To determine whether the prolonged 7-day incubation of T cells with MGD007 diminished their ability to support MGD007-mediated activity, T-cell–mediated responses were evaluated following re-exposure to MGD007 and freshly presented gpA33 target cells. As shown in Fig. 5D, T cells expanded following 7-day incubation with gpA33 target cells and MGD007, retained effective cytolytic activity when re-exposed to MGD007 and Colo205 (gpA33+) target cells even at a relatively low E:T ratio (1:1), with efficacy comparable with that supported by T cells incubated for 7 days with control DART (single-arm CD3) and gpA33 target cells. Interestingly however, evaluation of cytokine levels indicated that T cells pre-exposed for 7 days to MGD007 and gpA33 target cells were now inert in supporting IL-2, IFN-γ or TNF-α release. In contrast, the control-exposed T cells maintained cytokine response.

Figure 5.

MGD007 drives expansion of T cells with memory phenotype that retain cytolytic activity but reduced cytokine response. A, Proliferation of CFSE-labeled human T cells was monitored by flow cytometry upon co-culturing with gpA33+ LS174T cells (E:T = 10:1) in the presence of MGD007 (blue lines) or control DART (red lines and shaded) for 3 days (left) or 4 days (right). B, FACS analyses of freshly isolated T cells pre (blue) and 3 days post (red) co-culturing with gpA33+ Colo205 cells (E:T = 5:1) in presence of 0.4 μg/mL MGD007. CD25, PD-1, and LAG-3 surface marker expression determined on gated CD4+ and CD8+ T cells. Gray: unstained cells. C, FACS analyses of CD4+ and CD8+ T-cell subsets as defined by CCR7/CD45RA following 6-day co-culture of freshly isolated human T cells and gpA33+ SW948 colon cancer cells (E:T = 1:1) untreated or in the presence of MGD007 or control DART (both at 10 μg/mL). D, T cells collected following 6-day incubation with gpA33+ SW948 colon cancer cells in presence of control DART (top) or MGD007 (bottom) as described in (C) were re-cultured with Colo205-luc target cells (E:T = 1:1, 48 hours). Cytotoxicity levels were determined by decrease in luciferase fluorescence (left hand) whereas supernatants were evaluated for IL-2, IFN-γ, and TNF-α levels by ELISA.

Figure 5.

MGD007 drives expansion of T cells with memory phenotype that retain cytolytic activity but reduced cytokine response. A, Proliferation of CFSE-labeled human T cells was monitored by flow cytometry upon co-culturing with gpA33+ LS174T cells (E:T = 10:1) in the presence of MGD007 (blue lines) or control DART (red lines and shaded) for 3 days (left) or 4 days (right). B, FACS analyses of freshly isolated T cells pre (blue) and 3 days post (red) co-culturing with gpA33+ Colo205 cells (E:T = 5:1) in presence of 0.4 μg/mL MGD007. CD25, PD-1, and LAG-3 surface marker expression determined on gated CD4+ and CD8+ T cells. Gray: unstained cells. C, FACS analyses of CD4+ and CD8+ T-cell subsets as defined by CCR7/CD45RA following 6-day co-culture of freshly isolated human T cells and gpA33+ SW948 colon cancer cells (E:T = 1:1) untreated or in the presence of MGD007 or control DART (both at 10 μg/mL). D, T cells collected following 6-day incubation with gpA33+ SW948 colon cancer cells in presence of control DART (top) or MGD007 (bottom) as described in (C) were re-cultured with Colo205-luc target cells (E:T = 1:1, 48 hours). Cytotoxicity levels were determined by decrease in luciferase fluorescence (left hand) whereas supernatants were evaluated for IL-2, IFN-γ, and TNF-α levels by ELISA.

Close modal

Prolonged circulating half-life following repeat doses in cynomolgus monkeys

MGD007 pharmacokinetics were evaluated in cynomolgus monkeys administered 4 weekly doses of 30 or 100 μg/kg MGD007 by short intravenous infusion. Mean serum concentration–time profiles and PK parameters for 30 and 100 μg/kg MGD007 are shown in Fig. 6 and Supplementary Table S5. Cmax and AUC increased in proportion to dose, indicating linear PK. The mean clearance of MGD007 ranged from 0.7 to 0.8 mL/h/kg, lower than the glomerular filtration rate (GFR) in cynomolgus monkeys (∼125 mL/h/kg), indicating that MGD007 is not cleared by the kidney, as expected for a large molecular weight protein. Importantly, MGD007 demonstrated a prolonged mean beta half-life (t1/2,β) of 146 to 162 hours (6.1–6.8 days) and mean residence time (MRT) of 181–186 hours (7.5–7.8 days), consistent with an IgG Fc-bearing molecule. MGD007 was considered well tolerated in all animals treated at these dose levels, with no treatment-related increases in serum cytokine levels observed (IFN-γ, IL-2, IL-4, IL-5, IL-6, or TNF-α; Supplementary Fig. S5).

Figure 6.

MGD007 pharmacokinetic properties in non-human primates. MGD007 serum concentrations (mean ± SEM) following 30 or 100 μg/kg MGD007 administered intravenously over 2 hours once weekly for 4 weeks in cynomolgus monkeys (n = 4/sex/group).

Figure 6.

MGD007 pharmacokinetic properties in non-human primates. MGD007 serum concentrations (mean ± SEM) following 30 or 100 μg/kg MGD007 administered intravenously over 2 hours once weekly for 4 weeks in cynomolgus monkeys (n = 4/sex/group).

Close modal

Remarkable clinical responses have been observed in subsets of patients with cancer through therapeutic strategies leveraging the antitumor properties of T lymphocytes, including immune checkpoint inhibition (37), chimeric antigen receptor–expressing T cells (38) or bispecific molecules designed to co-engage T cells with cancer cells (8). A member of the latter category, the DART protein MGD007 was designed to co-engage T lymphocytes with colorectal cancer cells through the cell surface antigens, CD3 and gpA33, respectively, promote T-cell recruitment and antitumor activity.

The selection of the colorectal cancer targeting arm of MGD007 was based on a desire to target an antigen expressed on both the putative CSC and the differentiated malignant tumor cell populations. Recent studies have demonstrated the critical importance of the CSC population to tumor initiation, metastases and resistance to conventional therapies of colorectal cancer (39). Given their plasticity, CSC are not only able to differentiate into more mature “daughter” tumor cells, but may also switch back from daughter cells to CSC on cues from the tumor microenvironment niche (21, 22); therefore, eradication of both the CSC and the non-CSC populations appears necessary to ensure optimal therapeutic response and effectively curtail tumor progression.

To identify cell surface antigens expressed on the putative CSC, a panel of colorectal CSLC lines was developed. Various approaches have been previously used to isolate and characterize the CSC population from colorectal cancer, including direct cell sorting, tumor spheroid isolation and expansion, as well as organoid formation employing stem cell culturing media (17, 18, 40). Here, we have used a direct culturing method in defined media together with enrichment on a matrix system and without sorting. We previously employed a similar culturing strategy evolved from conditions developed for isolation and culturing of tissue stem cells, to develop clonal lung cancer-derived CSLC lines that exhibit properties of self-renewal, tumor initiation and differentiation (41). The panel of colorectal cancer CSLC lines we have currently developed covers a range of cancer stages and includes both KRAS WT and mutated specimens (Supplementary Table S2). Consistent with the characteristics ascribed to CSC, these lines can be maintained indefinitely, undergo a differentiation program under conditions that support tissue stem cell differentiation and, upon implantation in NOD-SCID mice, form tumors that morphologically match the histological features of the original cancer (Fig. 1A).

Whole-cell immunization with one of the CSLC lines yielded a panel of antibodies among which one mAb, RECA47, displayed the desired feature of homogeneous reactivity with the entire panel of CSLC lines (interestingly, the putative CSC markers, CD133 and CD44, displayed variable expression). The antigen, subsequently identified as gpA33, was found to be universally expressed across over 50 primary and metastatic colorectal cancer patient samples (Supplementary Table S3). The high penetrance and homogenous expression profile of gpA33 across patients with colorectal cancer observed with the RECA47 mAb is consistent with prior published studies (35). Indeed, gpA33 was originally defined as the target antigen for the A33 mAb, which was identified via a serological screen for antibodies that recognize colorectal cancer antigens (23). gpA33 is a 43 kDa membrane-bound glycoprotein comprising two immunoglobulin-like domains with homology to tight junction proteins CAR and JAM (36). Although the functional significance of gpA33 is unclear, studies in knockout mice indicate a role in colonic mucosal repair, consistent with its exclusive expression on normal human colon and small bowel epithelial cells (42).

Considering its attractiveness as a colon cancer antigen, several clinical studies evaluating antibody-based modalities targeting gpA33 have been attempted (23, 24, 43). Although initial studies with radiolabeled A33mAb were thwarted by immunogenicity and bone marrow toxicity associated with the radio-isotopes used, imaging analyses provided clear evidence of differential tumor retention and penetration compared with normal tissue (44). Several explanations for this selective targeting to tumors compared with normal tissue have been provided, but appeared on the basis of the more rapid turnover of gpA33 expressed by the colon epithelial cells compared with cancer cells (45). More recently, clinical evaluation of KRN330, a fully-human anti-gpA33 mAb, revealed initial evidence of clinical activity among patients with advanced and metastatic colorectal cancer, with effective tumor targeting at doses that were well tolerated (24). Unfortunately, KRN330 in combination with irinotecan failed to achieve significant clinical outcome in a subsequent study (46), suggesting the need for alternative gpA33 targeting modalities to yield benefit.

The appeal of T-cell–based therapies for cancer treatment, the high penetrance of gpA33 in colorectal cancer together with its expression by the putative CSC population and the prior clinical experience demonstrating selective tumor targeting by gpA33-antibody–based modalities all provided a strong rationale for the development of MGD007, a gpA33 x CD3-bispecific DART molecule that leverages T cells for colorectal cancer cell cytolysis. The DART platform has been previously demonstrated to support efficient redirected T-cell killing to target both hematological malignancies (9, 10, 32) and solid tumors (15). The compact nature of the DART molecule, as confirmed by its crystal structure (15), appears ideal for supporting optimal cell-cell association relative to other bispecific platforms (9). MGD007 is comprised of three polypeptide chains that self-assemble to provide monovalent binding to both gpA33 and CD3; it includes a modified IgG1 Fc domain to enhance its circulating half-life that is devoid of Fcγ receptor and complement binding. Flow-cytometry analyses confirmed the ability of MGD007 to bind natively expressed cell surface CD3 on human and cynomolgus monkey T cells and gpA33 expressed across a panel of human colorectal cancer cell lines or CHO cells engineered to express either the human or cynomolgus monkey gpA33.

Upon co-engagement with gpA33 on target cells, including model colorectal cancer CSLC lines, MGD007 mediates potent in vitro killing in the presence of T cells (Fig. 3); furthermore, MGD007 demonstrated antitumor activity in murine tumor xenograft models with gpA33-expressing human colorectal cancer cells co-implanted subcutaneously with human T cells, with doses ≥4 μg/kg resulting in significant tumor growth inhibition. Both CD8 and CD4 T cells can contribute to the killing, with enhanced activity observed with CD8 cells, consistent with prior reports and with the higher level of granzyme B/perforin of this subpopulation (Fig. 4). Considering the preponderance of Tregs in the tumor microenvironment (47), it is notable that MGD007 can also leverage a suppressive T-cell population for gpA33 target cell killing, an observation consistent with prior reports that CD3 directed bispecifics can leverage Tregs for cell-targeted lysis (48). As previously reported and consistent with the mechanism of action of CD3-based bispecific molecules, redirected T-cell killing of gpA33-positive tumor cells was accompanied by T-cell activation and concomitant cytokine production (Fig. 4A). Importantly, no cytokine release was observed with PBMC alone, attesting to both the absence of gpA33 expression on circulating immune cells and the lack of FcγR interaction by the crippled Fc domain. Considering that MGD007 was designed to maintain prolonged exposure through elevated pharmacokinetics, the effects of prolonged exposure to MGD007 on T-cell biology was also modelled in vitro. As anticipated, this led to T-cell expansion, with increases in the level of checkpoint inhibitor molecules, including PD-1 and LAG-3, and an effector-memory phenotype. Importantly, despite chronic exposure to MGD007, T cells still maintained significant killing but were drastically impaired in their ability to support a cytokine response, suggesting a potential for enhancing the cytolysis–v-cytokine release treatment window during the course of MGD007 exposure. These observations are indeed consistent with previous studies demonstrating that seven daily doses of a short serum half-life murine reactive EpCAM/CD3 bispecific BiTE (muS110) led to increased tolerability coinciding with reduced cytokines without compromise of effector function or antitumor activity (49).

Extended MGD007 serum half-life (∼1 week) was confirmed in cynomolgus monkeys, where both the CD3 (Fig. 2D) and gpA33 (Supplementary Fig. S2)–binding specificities are cross reactive. Upon administration of 4 weekly doses of either 30 or 100 μg/kg MGD007, serum concentrations were linear, with confirmed exposure throughout the 4-week dosing period. At these dose levels, no clinical symptoms, toxicity or increased circulating cytokine levels were observed. These dose levels far exceed the dose required to inhibit xenograft formation in mice (wherein toxicity analyses were not feasible due to lack of MGD007 reactivity to mouse gpA33 and CD3) but more importantly, Cmax levels observed at both dose levels were well above the concentration required to achieve maximal redirected T-cell killing of gpA33 expressing tumor cells in vitro. In subsequent studies, performed at exaggerated pharmacological doses of MGD007 (≥300 μg/kg) evidence of GI toxicity has been observed (J.G. Brown; unpublished observations) consistent with on-target engagement with gpA33 on intestinal cells.

Taken together, the high penetrance and uniformed expression of gpA33 in colorectal cancer, the potent lytic activity of MGD007 against gpA33+ colorectal cancer through leveraging of human T cells, including suppressive T-cell populations, and its favorable PK profile support the clinical evaluation of MGD007 in patients with colorectal cancer. A phase I dose-escalation and optimization study in advanced metastatic colorectal cancer is currently enrolling patients (NCT02248805).

R. Alderson is a director at In Vivo Modeling. S. Gorlatov is a scientist at Macrogenics. L. Liu has ownership interest (including stock, patents, etc.) in Stock option. All authors were employees of MacroGenics during course of study and as condition of employment receive stock options. No additional conflicts of interest were disclosed by authors.

Conception and design: P.A. Moore, K. Shah, R. Alderson, K.L. King, G.R. Chichili, L. Liu, S. Koenig, J. Mather, E. Bonvini, S. Johnson

Development of methodology: Y. Yang, R. Alderson, V. Long, D. Liu, J.C. Li, S. Burke, V. Ciccarone, K.L. King, P. Young, D. Loo, G.R. Chichili, L. Liu, F.Z. Chen, J. Mather, S. Johnson

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y. Yang, R. Alderson, J.C. Li, H. Li, J. Hooley, A. Easton, M. Licea, D. Loo, L. Liu, D.H. Smith, J.G. Brown

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): P.A. Moore, Y. Yang, R. Alderson, V. Long, D. Liu, J.C. Li, M. Licea, K.L. King, P. Young, D. Loo, G.R. Chichili, L. Liu, D.H. Smith, J.G. Brown, F.Z. Chen, E. Bonvini

Writing, review, and/or revision of the manuscript: P.A. Moore, R. Alderson, J.C. Li, A. Adami, G.R. Chichili, L. Liu, D.H. Smith, J.G. Brown, F.Z. Chen, S. Koenig, J. Mather, E. Bonvini, S. Johnson

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): P. Roberts, D. Liu, S. Gorlatov, A. Adami, D.H. Smith

Study supervision: P.A. Moore, F.Z. Chen, S. Koenig, E. Bonvini

Other (acquisition of data—antibody discovery and characterization): C.B. Fieger

Other (tumor study in animals and histology of tumor development): P. Young

We thank M. Lewis, C. Sung, and S. Sharma for help in article assembly and data analysis.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Marley
AR
,
Nan
H
. 
Epidemiology of colorectal cancer
.
Int J Mol Epidemiol Genet
2016
;
7
:
105
14
.
2.
Nosho
K
,
Baba
Y
,
Tanaka
N
,
Shima
K
,
Hayashi
M
,
Meyerhardt
JA
, et al
Tumour-infiltrating T-cell subsets, molecular changes in colorectal cancer, and prognosis: cohort study and literature review
.
J Pathol
2010
;
222
:
350
66
.
3.
Roelands
J
,
Kuppen
PJK
,
Vermeulen
L
,
Maccalli
C
,
Decock
J
,
Wang
E
, et al
Immunogenomic classification of colorectal cancer and therapeutic implications
.
Int J Mol Sci
2017
;
18
:
pii: E2229
.
4.
Ogino
S
,
Nosho
K
,
Irahara
N
,
Meyerhardt
JA
,
Baba
Y
,
Shima
K
, et al
Lymphocytic reaction to colorectal cancer is associated with longer survival, independent of lymph node count, microsatellite instability, and CpG island methylator phenotype
.
Clin Cancer Res
2009
;
15
:
6412
20
.
5.
Galon
J
,
Mlecnik
B
,
Bindea
G
,
Angell
HK
,
Berger
A
,
Lagorce
C
, et al
Towards the introduction of the ‘Immunoscore’ in the classification of malignant tumours
.
J Pathol
2014
;
232
:
199
209
.
6.
Le
DT
,
Uram
JN
,
Wang
H
,
Bartlett
BR
,
Kemberling
H
,
Eyring
AD
, et al
PD-1 blockade in tumors with mismatch-repair deficiency
.
N Engl J Med
2015
;
372
:
2509
20
.
7.
Frankel
SR
,
Baeuerle
PA
. 
Targeting T cells to tumor cells using bispecific antibodies
.
Curr Opin Chem Biol
2013
;
17
:
385
92
.
8.
Bargou
R
,
Leo
E
,
Zugmaier
G
,
Klinger
M
,
Goebeler
M
,
Knop
S
, et al
Tumor regression in cancer patients by very low doses of a T-cell–engaging antibody
.
Science
2008
;
321
:
974
7
.
9.
Moore
PA
,
Zhang
W
,
Rainey
GJ
,
Burke
S
,
Li
H
,
Huang
L
, et al
Application of dual affinity retargeting molecules to achieve optimal redirected T-cell killing of B-cell lymphoma
.
Blood
2011
;
117
:
4542
51
.
10.
Chichili
GR
,
Huang
L
,
Li
H
,
Burke
S
,
He
L
,
Tang
Q
, et al
A CD3xCD123 bispecific DART for redirecting host T cells to myelogenous leukemia: preclinical activity and safety in nonhuman primates
.
Sci Transl Med
2015
;
7
:
289ra82
.
11.
Al-Hussaini
M
,
Rettig
MP
,
Ritchey
JK
,
Karpova
D
,
Uy
GL
,
Eissenberg
LG
, et al
Targeting CD123 in acute myeloid leukemia using a T-cell–directed dual-affinity retargeting platform
.
Blood
2016
;
127
:
122
31
.
12.
Johnson
S
,
Burke
S
,
Huang
L
,
Gorlatov
S
,
Li
H
,
Wang
W
, et al
Effector cell recruitment with novel Fv-based dual-affinity re-targeting protein leads to potent tumor cytolysis and in vivo B-cell Depletion
.
J Mol Biol
2010
;
399
:
436
49
.
13.
Veri
MC
,
Burke
S
,
Huang
L
,
Li
H
,
Gorlatov
S
,
Tuaillon
N
, et al
Therapeutic control of B-cell activation via recruitment of Fcgamma receptor IIb (CD32B) inhibitory function with a novel bispecific antibody scaffold
.
Arthritis Rheum
2010
;
62
:
1933
43
.
14.
Liu
L
,
Lam
A
,
Alderson
R
,
Yang
Y
,
Li
H
,
Long
V
, et al
MGD011, humanized CD19 x CD3 DART protein with enhanced pharmacokinetic properties, demonstrates potent T-cell mediated anti-tumor activity in preclinical models and durable b-cell depletion in cynomolgus monkeys following once-a-week dosing [abstract]
.
In:
Proceedings of the 56th ASH Annual Meeting and Exposition; 2016 December 6–9
;
San Francisco, CA
:
ASH
; 
2016
.
15.
Root
AR
,
Cao
W
,
Li
B
,
LaPan
P
,
Meade
C
,
Sanford
J
, et al
Development of PF-06671008, a Highly Potent Anti-P-cadherin/Anti-CD3 Bispecific DART molecule with extended half-life for the treatment of cancer
.
Antibodies
52016
.
p
6
.
16.
Ricci-Vitiani
L
,
Lombardi
DG
,
Pilozzi
E
,
Biffoni
M
,
Todaro
M
,
Peschle
C
, et al
Identification and expansion of human colon-cancer-initiating cells
.
Nature
2007
;
445
:
111
5
.
17.
O'Brien
CA
,
Pollett
A
,
Gallinger
S
,
Dick
JE
. 
A human colon cancer cell capable of initiating tumour growth in immunodeficient mice
.
Nature
2007
;
445
:
106
10
.
18.
Fang
DD
,
Kim
YJ
,
Lee
CN
,
Aggarwal
S
,
McKinnon
K
,
Mesmer
D
, et al
Expansion of CD133(+) colon cancer cultures retaining stem cell properties to enable cancer stem cell target discovery
.
Br J Cancer
2010
;
102
:
1265
75
.
19.
Kreso
A
,
van Galen
P
,
Pedley
NM
,
Lima-Fernandes
E
,
Frelin
C
,
Davis
T
, et al
Self-renewal as a therapeutic target in human colorectal cancer
.
Nat Med
2014
;
20
:
29
36
.
20.
Junttila
MR
,
Mao
W
,
Wang
X
,
Wang
BE
,
Pham
T
,
Flygare
J
, et al
Targeting LGR5+ cells with an antibody-drug conjugate for the treatment of colon cancer
.
Sci Transl Med
2015
;
7
:
314ra186
.
21.
Vermeulen
L
,
Melo
DSE
,
van der
HM
,
Cameron
K
,
de Jong
JH
,
Borovski
T
, et al
Wnt activity defines colon cancer stem cells and is regulated by the microenvironment
.
Nat Cell Biol
2010
;
12
:
468
76
.
22.
Shimokawa
M
,
Ohta
Y
,
Nishikori
S
,
Matano
M
,
Takano
A
,
Fujii
M
, et al
Visualization and targeting of LGR5+ human colon cancer stem cells
.
Nature
2017
;
545
:
187
92
.
23.
Welt
S
,
Scott
AM
,
Divgi
CR
,
Kemeny
NE
,
Finn
RD
,
Daghighian
F
, et al
Phase I/II study of iodine 125-labeled monoclonal antibody A33 in patients with advanced colon cancer
.
J Clin Oncol
1996
;
14
:
1787
97
.
24.
Infante
JR
,
Bendell
JC
,
Goff
LW
,
Jones
SF
,
Chan
E
,
Sudo
T
, et al
Safety, pharmacokinetics and pharmacodynamics of the anti-A33 fully-human monoclonal antibody, KRN330, in patients with advanced colorectal cancer
.
Eur J Cancer
2013
;
49
:
1169
75
.
25.
Barnes
D
,
Sato
G
. 
Methods for growth of cultured cells in serum-free medium
.
Analytical Biochemistry
1980
;
102
:
255
70
.
26.
Roberts
PE
. 
Isolation and establishment of human tumor stem cells
.
Methods Cell Biol
2008
;
86
:
325
42
.
27.
Jhawer
M
,
Goel
S
,
Wilson
AJ
,
Montagna
C
,
Ling
YH
,
Byun
DS
, et al
PIK3CA mutation/PTEN expression status predicts response of colon cancer cells to the epidermal growth factor receptor inhibitor cetuximab
.
Cancer Res
2008
;
68
:
1953
61
.
28.
Smith
G
,
Carey
FA
,
Beattie
J
,
Wilkie
MJ
,
Lightfoot
TJ
,
Coxhead
J
, et al
Mutations in APC, Kirsten-ras, and p53–alternative genetic pathways to colorectal cancer
.
Proc Natl Acad Sci U S A
2002
;
99
:
9433
8
.
29.
Sato
T
,
Vries
RG
,
Snippert
HJ
,
van de Wetering
M
,
Barker
N
,
Stange
DE
, et al
Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche
.
Nature
2009
;
459
:
262
5
.
30.
Merlos-Suarez
A
,
Barriga
FM
,
Jung
P
,
Iglesias
M
,
Cespedes
MV
,
Rossell
D
, et al
The intestinal stem cell signature identifies colorectal cancer stem cells and predicts disease relapse
.
Cell Stem Cell
2011
;
8
:
511
24
.
31.
Loo
DT
,
Mather
JP
. 
Antibody-based identification of cell surface antigens: targets for cancer therapy
.
Curr Opin Pharmacol
2008
;
8
:
627
31
.
32.
Liu
L
,
Lam
CK
,
Long
V
,
Widjaja
L
,
Yang
Y
,
Li
H
, et al
MGD011, A CD19 x CD3 dual-affinity retargeting bi-specific molecule incorporating extended circulating half-life for the treatment of B-cell malignancies
.
Clin Cancer Res
2017
;
23
:
1506
18
.
33.
Sung
JA
,
Pickeral
J
,
Liu
L
,
Stanfield-Oakley
SA
,
Lam
CY
,
Garrido
C
, et al
Dual-affinity re-targeting proteins direct T cell-mediated cytolysis of latently HIV-infected cells
.
J Clin Invest
2015
;
125
:
4077
90
.
34.
Putnam
AL
,
Brusko
TM
,
Lee
MR
,
Liu
W
,
Szot
GL
,
Ghosh
T
, et al
Expansion of human regulatory T-cells from patients with type 1 diabetes
.
Diabetes
2009
;
58
:
652
62
.
35.
Garinchesa
P
,
Sakamoto
J
,
Welt
S
,
Real
F
,
Rettig
W
,
Old
L
. 
Organ-specific expression of the colon cancer antigen A33, a cell surface target for antibody-based therapy
.
Int J Oncol
1996
;
9
:
465
71
.
36.
Heath
JK
,
White
SJ
,
Johnstone
CN
,
Catimel
B
,
Simpson
RJ
,
Moritz
RL
, et al
The human A33 antigen is a transmembrane glycoprotein and a novel member of the immunoglobulin superfamily
.
Proc Natl Acad Sci U S A
1997
;
94
:
469
74
.
37.
Wolchok
JD
,
Kluger
H
,
Callahan
MK
,
Postow
MA
,
Rizvi
NA
,
Lesokhin
AM
, et al
Nivolumab plus ipilimumab in advanced melanoma
.
N Engl J Med
2013
;
369
:
122
33
.
38.
Grupp
SA
,
Kalos
M
,
Barrett
D
,
Aplenc
R
,
Porter
DL
,
Rheingold
SR
, et al
Chimeric antigen receptor-modified T cells for acute lymphoid leukemia
.
N Engl J Med
2013
;
368
:
1509
18
.
39.
Zeuner
A
,
Todaro
M
,
Stassi
G
,
De Maria
R
. 
Colorectal cancer stem cells: from the crypt to the clinic
.
Cell Stem Cell
2014
;
15
:
692
705
.
40.
Fujii
M
,
Shimokawa
M
,
Date
S
,
Takano
A
,
Matano
M
,
Nanki
K
, et al
A colorectal tumor organoid library demonstrates progressive loss of niche factor requirements during tumorigenesis
.
Cell Stem Cell
2016
;
18
:
827
38
.
41.
Mather
JP
,
Roberts
PE
,
Pan
Z
,
Chen
F
,
Hooley
J
,
Young
P
, et al
Isolation of cancer stem like cells from human adenosquamous carcinoma of the lung supports a monoclonal origin from a multipotential tissue stem cell
.
PLoS One
2013
;
8
:
e79456
.
42.
Pereira-Fantini
PM
,
Judd
LM
,
Kalantzis
A
,
Peterson
A
,
Ernst
M
,
Heath
JK
, et al
A33 antigen-deficient mice have defective colonic mucosal repair
.
Inflamm Bowel Dis
2010
;
16
:
604
12
.
43.
Welt
S
,
Ritter
G
,
Williams
C
 Jr
,
Cohen
LS
,
John
M
,
Jungbluth
A
, et al
Phase I study of anticolon cancer humanized antibody A33
.
Clin Cancer Res
2003
;
9
:
1338
46
.
44.
Scott
AM
,
Lee
FT
,
Jones
R
,
Hopkins
W
,
MacGregor
D
,
Cebon
JS
, et al
A phase I trial of humanized monoclonal antibody A33 in patients with colorectal carcinoma: biodistribution, pharmacokinetics, and quantitative tumor uptake
.
Clin Cancer Res
2005
;
11
:
4810
7
.
45.
Ackerman
ME
,
Chalouni
C
,
Schmidt
MM
,
Raman
VV
,
Ritter
G
,
Old
LJ
, et al
A33 antigen displays persistent surface expression
.
Cancer Immunol Immunother
2008
;
57
:
1017
27
.
46.
Bendell
JC
,
Lenz
HJ
,
Ryan
T
,
El-Rayes
BF
,
Marshall
JL
,
Modiano
MR
, et al
Phase 1/2 study of KRN330, a fully human anti-A33 monoclonal antibody, plus irinotecan as second-line treatment for patients with metastatic colorectal cancer
.
Invest New Drugs
2014
;
32
:
682
90
.
47.
Timperi
E
,
Pacella
I
,
Schinzari
V
,
Focaccetti
C
,
Sacco
L
,
Farelli
F
, et al
Regulatory T cells with multiple suppressive and potentially protumor activities accumulate in human colorectal cancer.
Oncoimmunology
2016
;
5
:
e1175800
.
48.
Choi
BD
,
Gedeon
PC
,
Herndon
JE
 II
,
Archer
GE
,
Reap
EA
,
Sanchez-Perez
L
, et al
Human regulatory T cells kill tumor cells through granzyme-dependent cytotoxicity upon retargeting with a bispecific antibody
.
Cancer Immunol Res
2013
;
1
:
163
.
49.
Amann
M
,
d'Argouges
S
,
Lorenczewski
G
,
Brischwein
K
,
Kischel
R
,
Lutterbuese
R
, et al
Antitumor activity of an EpCAM/CD3-bispecific BiTE antibody during long-term treatment of mice in the absence of T-cell anergy and sustained cytokine release
.
J Immunother
2009
;
32
:
452
64
.
50.
Moore
P
, et al
“Bi-specific monovalent diabodies that are capable of binding To gpA33 And CD3, and Uses Thereof
.”
International Patent Application No. WO 2015/026894
. February 
2015
.