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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.

Supplementary Table 1: Sixteen-locus STR analysis of tumor tissue and banked CSLC lines Supplementary Table 2: CSLC derivation and characteristics Supplementary Table 3: Immunohistochemical Analysis of Colorectal Cancer Tumor Specimens Supplementary Table 4: Equilibrium Dissociation Constants (KD) for Binding of MGD007 to Human CD3 or gpA33 Supplementary Table 5: MGD007 Pharmacokinetic Parameters Derived from Two-compartment Analysis in Cynomolgus Monkeys Supplementary Figure 1: In vitro CSLC differentiation in MatrigelTM 3D cultures Supplementary Figure 2: RECA47 mAb binding restricted to intestinal epithelium Supplementary Figure 3: MGD007 Flow Cytometry Analyses on Tumor Cell Lines Supplementary Figure 4: Expanded Treg population exhibits suppressive T-cell properties Supplementary Figure 5: Cytokine Levels in Monkeys Following Treatment with MGD007

PDF file, 81KB, Kinetic and Equilibrium binding constants, determined by surface plasmon resonance, for binding of murine BRCA84D, RES240 and MGA271 to human and cynomolgus monkey B7-H3.

PDF file, 75KB, Expression level of B7-H3 on ATCC cancer cell lines as determined by FLOW cytometry with anti-B7-H3 mAb BRCA84D. Solid curves represent isotype control staining; open curves represent staining with FI.

PDF file, 78KB, Measurement of the absolute level of circulating B cells (CD20+; Panel A), T cells (CD3+; Panel B) and NK cells (CD159a+)Panel C) in blood samples collected from cynomolgus monkeys at the indicated days prior to administration (Day -2), or following intravenous administration of PBS vehicle on Day 1 (all animals received PBS vehicle on Day 1) or MGA271 on Day 8 at the indicated dose. The panels on the left for each group represent all animals (N=6) in each dosing group, while the panels on the right represent only the recovery group animals (N=2) in each dosing group.

PDF file, 89KB, Measurement of IL-5 (Panel A), IL-6 (Panel B) and TNF-B (Panel C) cytokine levels in serum samples collected from cynomolgus monkeys prior to infusion (Pre) or at the indicated time points following intravenous administration of PBS vehicle (Day 1) or MGA271 at the indicated dose (Day 8). The top two panels of each cytokine data set represent all animals (N=6) in each dosing group, while the bottom three panels represent only the recovery group animals (N=2) in each dosing group. Sera from the samples were analyzed for cytokine levels using the Becton Dickinson Cytometric Bead Array Non-Human Primate Th1/Th2 Cytokine kit.

PDF file, 204KB, Panel A: Representative reactivity of anti-B7-H3 mAb BRCA84D with frozen tissue sections of prostate, breast, colon, lung, gastric and renal cancer. Panel B: Reactivity of anti-B7-H3 mAb BRCA69D on formalin-fixed paraffin embedded normal human spleen and lymph node tissues.

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Purpose: The goal of this research was to harness a monoclonal antibody (mAb) discovery platform to identify cell-surface antigens highly expressed on cancer and develop, through Fc optimization, potent mAb therapies toward these tumor-specific antigens.Experimental Design: Fifty independent mAbs targeting the cell-surface immunoregulatory B7-H3 protein were obtained through independent intact cell-based immunizations using human tissue progenitor cells, cancer cell lines, or cell lines displaying cancer stem cell properties. Binding studies revealed this natively reactive B7-H3 mAb panel to bind a range of independent B7-H3 epitopes. Immunohistochemical analyses showed that a subset displayed strong reactivity to a broad range of human cancers while exhibiting limited binding to normal human tissues. A B7-H3 mAb displaying exquisite tumor/normal differential binding was selected for humanization and incorporation of an Fc domain modified to enhance effector-mediated antitumor function via increased affinity for the activating receptor CD16A and decreased binding to the inhibitory receptor CD32B.Results: MGA271, the resulting engineered anti–B7-H3 mAb, mediates potent antibody-dependent cellular cytotoxicity against a broad range of tumor cell types. Furthermore, in human CD16A-bearing transgenic mice, MGA271 exhibited potent antitumor activity in B7-H3–expressing xenograft models of renal cell and bladder carcinoma. Toxicology studies carried out in cynomolgus monkeys revealed no significant test article-related safety findings.Conclusions: This data supports evaluation of MGA271 clinical utility in B7-H3–expressing cancer, while validating a combination of a nontarget biased approach of intact cell immunizations and immunohistochemistry to identify novel cancer antigens with Fc-based mAb engineering to enable potent antitumor activity. Clin Cancer Res; 18(14); 3834–45. ©2012 AACR.

PDF file, 59KB, Expanded normal tissue IHC analysis of B7-H3 on frozen tissues with MGA271 at optimal staining concentration (4 ug/ml) and at 5x the optimal concentration (20 ug/ml). The optimal staining concentration was determined by titration on the CaKi2 and Hs700T positive control cell lines to determine the lowest MGA271 concentration that yielded the greatest specific staining. (c) = cytoplasmic; (m) = membrane; (c,m) = cytoplasmic plus membrane.

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.

Introduction: A target-unbiased approach based on intact cell immunizations with fetal progenitor cells and cancer stem cells, followed by an immunohistochemistry (IHC) screen for cancer-specific candidates, led to the identification of anti-ADAM9 (a disintegrin and metalloproteinase) mAbs with highly differential tumor-versus-normal tissue binding. ADAM9 is a cell surface protein over-expressed in multiple tumors, with a possible role in promotion and progression of cancer through multiple mechanisms, including modulation of adhesion and migration as well as processing of tumorigenic and pro-angiogenic factors. In this preclinical study, we performed target/mAb validation and evaluated the therapeutic potential of anti-ADAM9 antibody-drug conjugates (ADCs) toward ADAM9-expressing solid cancers. Methods: IHC was performed with anti-ADAM9 mAbs to confirm and extend available data of human normal and tumor tissue expression. Epitope mapping studies were conducted to define epitope-specificity. mAbs were also screened to identify those that efficiently internalized into tumor cells. In vitro cellular processing studies were performed to further evaluate the mAbs as ADC candidates. Selected mAbs were converted to ADCs via chemical conjugation to potent anti-microtubule (DM4) or DNA alkylating (DGN549) agents; in vitro cytotoxicity studies were conducted with tumor cell lines representing human cancer types that overexpress ADAM9. A lead mAb was then selected for humanization and affinity maturation to yield a development candidate. Results: Anti-ADAM9 mAbs exhibited strong reactivity toward the tumor epithelium of solid cancers, including pancreatic, kidney, prostate, bladder, breast, colon, lung, and ovarian cancer, but limited reactivity toward normal tissues. Anti-ADAM9 mAbs were efficiently internalized and processed by tumor cell lines, including lines with only modest ADAM9 expression. Anti-ADAM9 ADCs exhibited specific, dose-dependent cytotoxicity toward ADAM9-positive cancer cell lines in vitro, with IC50 values in the sub-nanomolar range. Humanization and affinity maturation of the lead mAb yielded a development candidate that retains potent antitumor activity toward ADAM9-positive tumor cell lines and equivalent, high affinity binding to both human and cynomolgus monkey ADAM9. Conclusion: ADAM9 is a cell surface antigen that is over-expressed on a wide range of solid cancers. Anti-ADAM9 mAbs that were strongly reactive with representative tumors exhibited high affinity for the antigen and were efficiently internalized and processed by ADAM9-bearing tumor cells. Anti-ADAM9 ADCs demonstrated dose-dependent cytotoxicity in vitro toward a panel of ADAM9-positive tumor cell lines. Our findings demonstrate that an ADC targeting ADAM9 may serve as a potential therapeutic for ADAM9-expressing solid tumors. Citation Format: Juniper A. Scribner, Bhaswati Barat, Stuart W. Hicks, Nicholas C. Yoder, Thomas Son, Lusiana Widjaja, Gundo Diedrich, Sergey Gorlatov, Jeff Hooley, Ann Easton, Peter Lung, Anushka De Costa, Francine Chen, Michael Chiechi, Pam Li, Monica Licea, Timothy E. Hotaling, Michael Spliedt, Valentina Ciccarone, Nadia Gantt, James Tamura, Megan E. Fuller, Molly McShea, Scott Koenig, Syd Johnson, Paul A. Moore, Ezio Bonvini, Deryk Loo. Target validation, antibody discovery and preclinical data supporting ADAM9 as an antibody-drug conjugate therapeutic target for solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 38. doi:10.1158/1538-7445.AM2017-38

Introduction: The combination of monoclonal antibodies (mAbs) that targets the immune checkpoint molecules CTLA-4 and PD-1 has shown clinical benefit beyond that observed with either mAb alone. This finding has prompted exploring whether such an approach could be applied within the context of additional combinations of checkpoint molecules, such as PD-1 and lymphocyte activation gene-3 (LAG-3). Animal tumor models have validated combining anti-PD-1 with anti-LAG-3 mAbs in eliciting synergistic tumor-eradicating immunity; expression of PD-1 and LAG-3 on exhausted T cells and tumor infiltrating lymphocytes (TILs) further supports their dual-targeting. We have developed a bispecific DART® protein that targets PD-1 and LAG-3, aimed at inducing potent antitumor immunity through simultaneous blockade of non-redundant checkpoint pathways intrinsic to exhausted T cells. Methods: mAbs against PD-1 and LAG-3 were generated and selected for DART conversion based on binding, biophysical and functional blocking against their respective receptor/ligand axes and functional activity in re-activation of prior superantigen-stimulated T cells or in antigen-specific recall assays. Results: Lead PD-1 and LAG-3 mAbs demonstrating favorable functional properties were selected for humanization. Immunohistochemistry confirmed that the lead LAG-3 and PD-1 mAbs display restricted lymphocyte expression in human tissues and overlapping expression in TILs. The humanized mAbs were assembled into MGD013, an Fc-bearing PD-1 x LAG-3 DART protein that demonstrated favorable biophysical and manufacturability properties. MGD013 bound specifically with high affinity to PD-1 and LAG-3 as well as to target-expressing cell lines and chronically-activated T cells. MGD013 blocked PD-1/PD-L1, PD-1/PD-L2 and LAG-3/HLA (MHC-II) interactions and PD-1 signaling. Further functional characterization of MGD013 revealed enhanced cytokine secretion in response to antigenic re-challenge of previously stimulated T cells compared to that observed upon independent blockade of either the PD-1 or LAG-3 pathways alone. Furthermore, under the above experimental conditions, MGD013 mediated greater cytokine secretion than that observed with the combination of equivalent (equimolar) levels of replicas of the approved PD-1 mAb, nivolumab, and the LAG-3 mAb, 25F7, which is currently undergoing clinical testing. Finally, cynomolgus monkey pharmacokinetic studies demonstrated a prolonged circulating half-life consistent with that of an Fc-bearing molecule. Conclusions: MGD013 blocks both PD-1 and LAG-3 pathways, resulting in enhanced T-cell responses compared to single or combination mAb blockade. Together with favorable cynomolgus monkey PK, these studies support further clinical development of MGD013. Citation Format: Ross LaMotte-Mohs, Kalpana Shah, Doug Smith, Sergey Gorlatov, Valentina Ciccarone, James Tamura, Hua Li, Jill Rillema, Monica Licea, Leilei He, Farha Vasanwala, Wei Chen, Xiao-Tao Yao, Francine Chen, Jennifer Brown, Jeffrey Nordstrom, Scott Koenig, Ezio Bonvini, Syd Johnson, Paul Moore. MGD013, a bispecific PD-1 x LAG-3 Dual-Affinity Re-Targeting (DART®) protein with T-cell immunomodulatory activity for cancer treatment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3217.

Introduction. IL13Rα2 is a membrane-bound protein expressed on a number of malignant tumors, but not at significant levels in most normal tissues. Clinically tested therapies to treat cancer by targeting IL13Rα2 to date have included a ligand toxin conjugate (IL13-PE) and IL13Rα2 directed CAR-T cells. Both strategies appear to be well tolerated, although with mixed results in tumor eradication. An alternative strategy for targeting IL13Rα2 is an IL13Rα2 x CD3 Dual-Affinity Re-Targeting (DART®) bispecific molecule designed to co-engage IL13Rα2 on tumor cells and cytotoxic T cells through CD3, resulting in killing of tumor cells. Methods. Mouse monoclonal antibodies (mAbs) were generated using standard immunization protocols followed by binding and epitope binning analyses. Cell binding was performed by flow cytometry and frozen normal and tumor tissues analyses by immunohistochemistry (IHC). In vitro studies were performed with cancer cell lines and primary human T cells or peripheral blood mononuclear cells (PBMCs). In vivo studies were performed in immune-deficient tumor-bearing mice co-implanted with activated human T cells or reconstituted with human PBMCs. Results. A panel of mAbs that bind human and cynomolgus monkey IL13Rα2 proteins were selected, representing a range of binding characteristics and epitope diversity. IHC showed favorable normal vs tumor tissue reactivity, with high levels of IL13Rα2 expression in glioblastoma and melanoma. The mAb panel was converted to DART molecules with an anti-CD3 arm, assessed for the ability to mediate cytotoxic T lymphocyte (CTL) activity and cytokine release and a lead selected for humanization and engineering into an IL13Rα2 x CD3 DART molecule incorporating an Fc domain to prolong circulating half-life. The IL13Rα2 x CD3 DART protein bound human and cynomolgus IL13Rα2 (KD = 0.64 nM and 0.5 nM, respectively) and mediated CTL activity against LOX-IMVI (melanoma) and SK-MES-1 (lung adenocarcinoma) target cells. Cytokines (IFN-γ, IL-10, and TNF-α) were released in the presence of IL13Rα2-expressing target cells and human PBMC, but not with PBMCs alone. Administration of the IL13Rα2 x CD3 DART molecule (≤50 μg/kg IV for 4 consecutive days) prevented tumor growth when tumor cells (A375 and LOX-IMVI melanoma, U87 glioblastoma, or DAOY medulloblastoma cell lines) were co-mixed with activated human T cells and implanted subcutaneously in NOD/SCID/IL2gamma-chain null mice. Tumor eradication was also observed in NOD/SCID/IL2gamma-chain/MHCI KO mice reconstituted with human PBMCs and bearing established LOX-IMVI melanoma tumors following 2 weekly IV doses of 50 μg/kg IL13Rα2 x CD3 DART molecule. Conclusions: Robust in vitro and in vivo antitumor activity for an IL13Rα2 x CD3 DART protein was demonstrated. Additional studies are underway to further characterize the molecule as a development candidate for treatment of IL13Rα2-positive cancers. Citation Format: Jill Rillema, Monica Licea, Shereen Saini-Lal, Doug Smith, Francine Chen, Annie Lam, Yinhua Yang, Liqin Liu, James Tamura, Ralph Alderson, Ezio Bonvini, Syd Johnson, Paul Moore. Development of an IL13Ralpha2 x CD3 bispecific DART® protein for redirected T-cell killing of solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1498.

Introduction: Recent studies have implied that solid tumors including colon cancer, can arise from tissue-specific stem cells. Defined serum-free culture conditions, employed to select for fetal colon epithelial progenitor cells, were utilized to isolate and expand a sub-population of epithelial cells from human tumors resected from a repertoire of colon cancer patients (Roberts 2011, AACR #5211). These cancer stem-like cell (CSLC) lines are stable homogenous populations that, when cultured under conditions that promote colon crypt differentiation, differentiate into organoids containing the 3 principal cell types seen in the colon and in differentiated colorectal tumors. In vivo they form tumors that fully recapitulate the morphologic and phenotypic characteristics of the patients’ original tumors. While any consistent definition of cancer stem cell properties remains elusive, these CSLC providing unique tools to interrogate the biology of CSC or tumor initiating cells, and identify CSC-expressed surface antigens. Methods: Cell immunizations were performed with colon CSLC. mAbs were analyzed by IHC to determine their reactivity toward normal and cancer tissues as well as a panel of cancer lines including colon CSLC. Antitumor bioactivity screens were followed by antigen identification of promising candidates. Affymetrix U133 micro-array analysis was performed to profile gene expression and Taqman analyses performed to identify differentially expressed genes compared to non-CSLC control populations. Results: mAbs from CSLC immunizations included a subset that displayed uniform binding across the complete panel of CSLC lines while displaying high penetrance of expression on both primary and metastatic colon cancer tissues. Expression cloning identified the antigen as glycoprotein A33 (gpA33), which was confirmed by IP and SPR analyses. Transcriptional profiling confirmed elevated gpA33 expression across the panel of colon CSLC in addition to revealing enhanced LGR5, ASCL2 and SOX9 expression, consistent with the CSLC having a colon stem cell origin. The uniform expression of gpA33 on putative cancer stem cell populations is distinguished from other cell surface molecules generally regarded as “canonical” CSC markers, including CD133 and CD44, which are variably expressed. Conclusion: gpA33 is an antigen with excellent expression penetrance in colon cancer and colon cancer-derived CSLC. As shown here, immunizations with CSLC lines can yield antibodies specific for colon cancer antigens that may also represent CSC expressed markers. This provides an example how new therapeutic antibody candidates can be generated to recognize and target both differentiated cancer cells as well as their associated CSC population. Citation Format: Jonathan Li, Claudia B. Fieger, Penny Roberts, Doug Smith, Monica Licea, Jeff Hooley, Francine Chen, Kathy King, Jennie Mather, Ezio Bonvini, Deryk Loo, Paul A. Moore. Identification of gpA33 as a marker expressed on colon cancer stem cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3763. doi:10.1158/1538-7445.AM2013-3763

Antigens that are tumor-specific or over-expressed on cancer cells represent opportunities for developing target-specific antibody-based therapeutics. Utilizing a non-target-biased intact cell-based immunization approach, we have generated greater than 1600 mAbs to cell-surface proteins that are expressed on cancer cells. A subset of the mAbs exhibit differential reactivity to tumor cells compared to normal cells. One of the differentially expressed proteins identified was B7-H3 (also referred to as CD276). B7-H3 is a member of the B7 family of immune regulatory proteins. Over-expression of B7-H3 has been correlated with disease severity and outcome in a growing number of cancer types, and several lines of evidence support a functional role for B7-H3 in cancer. In this report we describe the development of a humanized anti-B7-H3 mAb, designated MGA271, which bears an engineered Fc domain that imparts enhanced antibody-dependent cellular cytotoxicity (ADCC) through increased affinity for the human activating Fc-gamma receptor IIIA (CD16A) and decreased affinity for the human inhibitory Fc-gamma receptor IIB (CD32B). MGA271 displays strong reactivity to multiple tumors including kidney, prostate, pancreatic, breast, colon, gastric and ovarian cancer as well as melanoma, but limited reactivity toward normal human tissues. Independently of the donor's CD16A genotype, MGA271 mediates ADCC in vitro against human tumor cell lines representing these tumor types, as well as to human lung and gastrointestinal cell lines that exhibit properties of cancer stem cells. MGA271 exhibits potent anti-tumor activity toward B7-H3-expressing tumor xenografts in mice knocked-out for the murine CD16 gene and transgenic for the low affinity allele of human CD16A. Single- and repeat-dose toxicology studies were carried out in cynomolgus monkeys and no significant test article-related safety findings were observed. Taken together, these data support the clinical development of MGA271 for the treatment of patients who have B7-H3-expressing cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2725. doi:1538-7445.AM2012-2725