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ADAM metallopeptidase domain 9 (ADAM9) is a member of the ADAM family of multifunctional, multidomain type 1 transmembrane proteins. ADAM9 is overexpressed in many cancers, including non–small cell lung, pancreatic, gastric, breast, ovarian, and colorectal cancer, but exhibits limited expression in normal tissues. A target-unbiased discovery platform based on intact tumor and progenitor cell immunizations, followed by an IHC screen, led to the identification of anti-ADAM9 antibodies with selective tumor-versus-normal tissue binding. Subsequent analysis revealed anti-ADAM9 antibodies were efficiently internalized and processed by tumor cells making ADAM9 an attractive target for antibody–drug conjugate (ADC) development. Here, we describe the preclinical evaluation of IMGC936, a novel ADC targeted against ADAM9. IMGC936 is comprised of a high-affinity humanized antibody site-specifically conjugated to DM21-C, a next-generation linker-payload that combines a maytansinoid microtubule-disrupting payload with a stable tripeptide linker, at a drug antibody ratio of approximately 2.0. In addition, the YTE mutation (M252Y/S254T/T256E) was introduced into the CH2 domain of the antibody Fc to maximize in vivo plasma half-life and exposure. IMGC936 exhibited cytotoxicity toward ADAM9-positive human tumor cell lines, as well as bystander killing, potent antitumor activity in human cell line-derived xenograft and patient-derived xenograft tumor models, and an acceptable safety profile in cynomolgus monkeys with favorable pharmacokinetic properties. Our preclinical data provide a strong scientific rationale for the further development of IMGC936 as a therapeutic candidate for the treatment of ADAM9-positive cancers. A first-in-human study of IMGC936 in patients with advanced solid tumors has been initiated (NCT04622774).

5000 Hs700T parental cells only in the presence of 6.7 nM MGC018

5000 Hs700T B7-H3 KO/RFP cells alone in the presence of 6.7 nM MGC018

Supplementary Data

Co-culture of 5000 Hs700T parental cells and 5000 Hs700T B7-H3 KO/RFP cells in the presence of 6.7 nM MGC018

B7-H3, also referred to as CD276, is a member of the B7 family of immune regulatory proteins. B7-H3 is overexpressed on many solid cancers, including prostate cancer, renal cell carcinoma, melanoma, squamous cell carcinoma of the head and neck, non–small cell lung cancer, and breast cancer. Overexpression of B7-H3 is associated with disease severity, risk of recurrence and reduced survival. In this article, we report the preclinical development of MGC018, an antibody–drug conjugate targeted against B7-H3. MGC018 is comprised of the cleavable linker-duocarmycin payload, valine-citrulline-seco duocarmycin hydroxybenzamide azaindole (vc-seco-DUBA), conjugated to an anti-B7-H3 humanized IgG1/kappa mAb through reduced interchain disulfides, with an average drug-to-antibody ratio of approximately 2.7. MGC018 exhibited cytotoxicity toward B7-H3–positive human tumor cell lines, and exhibited bystander killing of target-negative tumor cells when cocultured with B7-H3–positive tumor cells. MGC018 displayed potent antitumor activity in preclinical tumor models of breast, ovarian, and lung cancer, as well as melanoma. In addition, antitumor activity was observed toward patient-derived xenograft models of breast, prostate, and head and neck cancer displaying heterogeneous expression of B7-H3. Importantly, MGC018 exhibited a favorable pharmacokinetic and safety profile in cynomolgus monkeys following repeat-dose administration. The antitumor activity observed preclinically with MGC018, together with the positive safety profile, provides evidence of a potentially favorable therapeutic index and supports the continued development of MGC018 for the treatment of solid cancers.

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

Supplementary Data from Preclinical Evaluation of IMGC936, a Next-Generation Maytansinoid-based Antibody–drug Conjugate Targeting ADAM9-expressing Tumors

Supplementary Materials and Methods

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.

B7-H3, also referred to as CD276, is a member of the B7 family of immune regulatory proteins. B7-H3 is overexpressed on many solid cancers, including prostate cancer, renal cell carcinoma, melanoma, squamous cell carcinoma of the head and neck, non–small cell lung cancer, and breast cancer. Overexpression of B7-H3 is associated with disease severity, risk of recurrence and reduced survival. In this article, we report the preclinical development of MGC018, an antibody–drug conjugate targeted against B7-H3. MGC018 is comprised of the cleavable linker-duocarmycin payload, valine-citrulline-seco duocarmycin hydroxybenzamide azaindole (vc-seco-DUBA), conjugated to an anti-B7-H3 humanized IgG1/kappa mAb through reduced interchain disulfides, with an average drug-to-antibody ratio of approximately 2.7. MGC018 exhibited cytotoxicity toward B7-H3–positive human tumor cell lines, and exhibited bystander killing of target-negative tumor cells when cocultured with B7-H3–positive tumor cells. MGC018 displayed potent antitumor activity in preclinical tumor models of breast, ovarian, and lung cancer, as well as melanoma. In addition, antitumor activity was observed toward patient-derived xenograft models of breast, prostate, and head and neck cancer displaying heterogeneous expression of B7-H3. Importantly, MGC018 exhibited a favorable pharmacokinetic and safety profile in cynomolgus monkeys following repeat-dose administration. The antitumor activity observed preclinically with MGC018, together with the positive safety profile, provides evidence of a potentially favorable therapeutic index and supports the continued development of MGC018 for the treatment of solid cancers.

The transcription factor nuclear factor kappaB (NF-kappaB) regulates the expression of both anti-apoptotic and proapoptotic genes. Death receptor 5 (DR5, TRAIL-R2) is a proapoptotic protein considered to be a potential target for cancer therapy, and its expression is mediated by NF-kappaB. The mechanism of NF-kappaB-induced DR5 expression is, however, unknown. Herein, we determined that etoposide-induced DR5 expression requires the first intronic region of the DR5 gene. Mutation of a putative NF-kappaB binding site in this intron eliminates DR5 promoter activity, as do mutations in the p53 binding site in this region. Reduction in p53 expression also blocks p65 binding to the intronic region of the DR5 gene, indicating cooperation between p53 and p65 in DR5 expression. In contrast, the anti-apoptotic stimulus, epidermal growth factor (EGF), fails to increase DR5 expression but effectively activates NF-kappaB and induces p65 binding to the DR5 gene. EGF, however, induces the association of histone deacetylase 1 (HDAC1) with the DR5 gene, whereas etoposide treatment fails to induce this association. Indeed, HDAC inhibitors activate NF-kappaB and p53 and upregulate DR5 expression. Blockage of DR5 activation decreased HDAC inhibitor-induced apoptosis, and a combination of HDAC inhibitors and TRAIL increased apoptosis. This provides a mechanism for regulating NF-kappaB-mediated DR5 expression and could explain the differential roles NF-kappaB plays in regulating apoptosis.

The preclinical safety assessment program of a drug product is determined by whether it is a pharmaceutical (small molecule) or biopharmaceutical (large molecule). Each has its own set of regulatory requirements that continue to advance over time. In particular, the biopharmaceutical program has rapidly evolved as the investigation of the number of products increases and knowledge accumulates. This chapter is an overview of the differences and similarities in the preclinical programs between the two drug classes and the requirements to move into clinical testing.

One of the challenges in cancer therapy is to eradicate tumor cells while minimizing the toxic side effects to normal tissue that can rapidly become dose-limiting. In this regard, the unique specificity of antibodies enables the targeting of antigens that are differentially or aberrantly expressed on tumor cells while ignoring their normal counterparts [1]. To date, six IgG antibodies have received FDA approval for the treatment of cancer, Herceptin (Trastuzumab), Rituxan (Rituximab), Avastin (Bevacizumab), Campath (Alemtuzumab), Erbitux (Cetuximab), and Vectibix (Panitumumab), and all have shown varying degrees of clinical and commercial success [2]. While designed to target tumor cells with nanomolar affinity, clinical evidence would suggest that the anticancer mechanisms mediated by these antibodies are not on their own sufficient to provide a prolonged clinical benefit [3].To that end, other strategies have been explored to enhance antibody potency while still exploiting their targeting function. One such approach has been to attach a cytotoxic payload to an antibody that when delivered to a cancer cell induces a highly potent cell death signal [4]. The most common payloads attached to antibodies or antibody fragments are small molecule drugs, radionucleotides, and toxins [1, 5–8]. Two radionucleotide-conjugated antibodies Zevalin (Ibritumomab tiuxetan) and Bexxar (Tositumomab-/I131) and one antibiotic-conjugated antibody Mylotarg (Gemtuzumab Ozogamicin) have been approved, although Mylotarg was subsequently withdrawn [9]. In addition, Ontak a diptheria toxin (DT) conjugated to an IL2 cytokine received approval for the treatment of cutaneous T cell lymphoma [10]. A variety of antibody–drug conjugates (ADCs) such as the anti-HER2 trastuzumab-DM-1 are currently being evaluated in the clinic as antibody conjugates have proven themselves superior to the naked antibody in xenograft tumor model [11]. Similarly, a variety of immunotoxins have been evaluated in the clinic, but as yet none have received FDA approval; however, those targeting leukemic cancers such as BL22, an anti-CD22 dsFv linked to truncated Pseudomonas exotoxin A (ETA), have been particularly successful [12, 13].

VB6-845 is a recombinant immunotoxin comprised of deBouganin (a de-immunized plant toxin) genetically linked to an epithelial cell adhesion molecule (EpCAM)-targeting humanized Fab fragment (4D5MOCB). EpCAM is highly expressed on a wide range of epithelial tumors but has limited expression on most normal epithelia and therefore represents an excellent target for immunotherapy. A comprehensive preclinical evaluation was performed to determine the safety and suitability of VB6-845 as a systemically administered drug for the treatment of solid tumors. Efficacy studies in mice demonstrated that VB6-845 specifically and potently targeted EpCAM-positive tumors. In a dose-ranging study in Sprague-Dawley rats, single doses of VB6-845 were well-tolerated resulting in a no-observable adverse effect level (NOAEL) of 100 mg/kg whereas repeated doses of VB6-845 resulted in vascular leak-associated symptoms particularly at higher dose levels. However, much higher doses in Cynomolgus monkeys were well-tolerated when given as a 3-hour infusion mimicking the intended route of administration in the clinic. In addition, VB6-845 proved to be minimally immunogenic in monkeys. The toxicological data obtained in Cynomolgus monkeys indicated an excellent safety profile with a NOAEL value of 30 mg/kg (equivalent to a 10 mg/kg dose in humans). These results are supportive of an exploratory Phase I trial.

The clinical development of immunotoxins in the treatment of solid tumors has been impeded in part, by the induction of an immune response directed primarily against the toxin moiety. Bouganin, a type I ribosome inactivating protein isolated from the leaf of Bougainvillea spectabilis Willd, was mutated to remove the T-cell epitopes while preserving the biological activity of the wild-type molecule. The T-cell epitope-depleted variant of bouganin (de-bouganin) was genetically linked to an anti-epithelial cell adhesion molecule (EpCAM) Fab moiety via a peptidic linker containing a furin proteolytic site to create the fusion construct VB6-845. To determine the optimal construct design for VB6-845, several dicistronic units where de-bouganin was genetically linked to either the N-terminal or C-terminal of either the heavy or light chain were engineered. Only the C-terminal variants expressed the full-length molecule. An in vitro assessment of the biological activity of VB6-845 showed that it bound and selectively killed EpCAM-positive cell lines with a greater potency than many commonly used chemotherapeutic agents. In vivo efficacy was demonstrated using an EpCAM-positive human tumor xenograft model in SCID mice with the majority of the mice treated being tumor free at the end of the study.

Immunotoxins continue to be actively investigated as viable alternatives to conventional therapies for a variety of diseases. An array of different recombinant, antibody formats have improved the overall in vitro and preclinical in vivo efficacy of immunotoxins. This article describes the development of immunotoxins and the preclinical development required for advancing VB4-845, an anti–EpCAM targeting scFv linked to a truncated form of Pseudomonas exotoxin A(252-608), into the clinic. Keywords: immunotoxins; antibody affinity; immunogenicity; cytotoxicity; safety assessment

Background: Ado-trastuzumab emtansine (T-DM1), a recently approved antibody-drug conjugate (ADC), is comprised of the monoclonal antibody trastuzumab linked to the cytotoxic agent mertansine. Trastuzumab targets and inhibits growth of HER2/neu-receptor expressing tumors and mertansine enters and kills the cells by binding to tubulin. Trastuzumab alone is sufficient to effectively treat high (3+) HER2-expressing breast cancers; however, whether T-DM1 is effective in lower HER2-expressing cancers is unclear. To better understand how HER-2 expression correlates with activity of trastuzumab or T-DM1 we characterized HER2 expression in our START-PDX breast panel and screened trastuzumab and T-DM1 efficacy in each model. Study endpoints included tumor volume and time from treatment initiation. Reported results included tumor growth inhibition or delay and regression. Methods: Methods: START-PDX breast models were established in immune-deficient mice from primary or metastatic patient tissue and once established were confirmed by histologic comparative analysis and linked with patient treatment and outcome data. For each model, DNA was extracted and subjected to exon sequencing of 207 known oncogenes; growth factor receptor and ligand densities were interrogated using immunohistochemistry and quantitative RNA in situ hybridization. Drug sensitivity studies were performed evaluating sensitivity of models to single agent trastuzumab and T-DM1, administered for at least four weeks. Study endpoints included tumor volume and time from treatment initiation with tumor growth inhibition, delay and regression reported at study completion. Results: HER2 or HER3 and comparative heregulin expression occurred in 67% of models. EGFR expression was reported in 50% of models Efficacy studies revealed T-DM1 but not trastuzumab was active both in several high- and low-HER2 expressing models. Interestingly, models reporting higher T-DM1 sensitivity had no detectable HER3 expression. For example ST996 and ST910 showed similar HER2 and EGFR expression but ST996 (HER3 3+) was insensitive to T-DM1 whereas ST910 (HER3 0+) reported significant tumor growth inhibition compared with control. Conclusion: We have established a panel of breast PDX models and characterized mutation status, receptor and ligand density and drug sensitivity. We found T-DM1 active towards a number of breast PDX models even with low HER2 expression. HER3 co-expression may serve as a negative predictor for T-DM1 activity. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C104. Citation Format: Michael J. Wick, Jennifer Brown, Teresa L. Vaught, Justin Meade, Anthony W. Tolcher, Drew Rasco, Amita Patnaik, Amy Lang, Murali Beeram, Kyriakos P. Papadopoulos. Preclinical sensitivity of low and high-HER2 expressing START-PDX breast models to T-DM1 and effects of HER3 expression on drug antitumor activity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C104.

Background: Recent FDA approval of B-Raf and Mek inhibitors has improved treatment outcomes for patients with BRAFV600E/K metastatic melanoma. However, most patients receiving these agents progress within a year and are left with limited treatment options. Few validated screening tools exist to evaluate potentially beneficial therapies for these resistant/refractory patients. To address this deficiency we have established patient-derived xenograft (PDX) model sets by collecting serial samples at timepoints when patients demonstrated sensitivity and resistance to B-Raf and Mek inhibitors and characterized each by mutation, receptor and ligand density and drug sensitivity. Methods: Paired, sensitive/insensitive PDX melanoma models from three patients were established in athymic nude mice from primary or metastatic patient tissue. Baseline samples for each paired model were collected from patients who subsequently reported clinical response to a therapy and again once the patient progressed on that therapy. Established models were confirmed by histologic comparative analysis and linked with patient treatment and outcome data. For each model DNA was extracted and subjected to exon sequencing of 207 known oncogenes; growth factor receptor and ligand densities were interrogated using immunohistochemistry and quantitative RNA in situ hybridization. Drug sensitivity studies were performed evaluating and comparing each model following treatment with oral vemurafenib, trametinib or dabrafenib. Study endpoints included tumor volume and time from treatment initiation, with tumor growth inhibition, delay and regression reported at study completion. Results: The B-RafV600E models ST052, ST052B, ST052C and ST052D reported sensitivity or resistance to vemurafenib correlative with clinical response and progression. Trametinib but not vemurafenib was active towards the ST361 B-RafV600R model while the ST361B model was found insensitive to both agents. Trametinib sensitivity to the ST697 and ST697B H-RasQ61K models was correlative with clinical response and progression. Dabrafenib sensitivity was similar to vemurafenib in evaluated models. Expression of HER3, a mechanism of possible adaptive resistance to targeted therapies, but not EGFR or HER2 was reported in all models and was relatively unchanged in serial models sets; heregulin expression was also determined in each model. Conclusion: We have established patient-derived xenograft (PDX) model sets with sensitivity and resistance to B-Raf and Mek inhibitors correlative with clinical response and progression. Receptor and ligand expression was identified and maintained in serial models. Based on these results this panel can serve as a valuable screening tool to evaluate potentially beneficial therapies for patients resistant/refractory to B-Raf and Mek inhibitors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C10. Citation Format: Michael J. Wick, Justin Meade, Jennifer Brown, Teresa L. Vaught, Anthony W. Tolcher, Lizette Gamez, Drew Rasco, Amita Patnaik, Ronald Drengler, Kyriakos P. Papadopoulos. Establishment and characterization of melanoma patient-derived xenograft (PDX) model sets: correlation with clinical response and progression using serial biopsy sampling. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C10.

The specificity, detection limit, and stability of twelve anti-Salmonella monoclonal antibodies (Mabs) were evaluated by cloth-based enzyme immunoassay (CEIA) and polymyxin-cloth based enzyme immunoassay (p-CEIA). Using the p-CEIA, five Mabs were found to react with cholate extracted lipopolysaccharide (LPS) antigens of all 44 Salmonella strains representing 19 different serogroups examined, with the exception of the one strain of serogroup-O tested. These five Mabs did not react with cholate extracts of any of 16 Gram-positive or Gram-negative non-Salmonella bacteria tested. The detection limit of purified S. typhimurium LPS antigen in the p-CEIA was approximately 40 ng for four of the Mabs and approximately 20 ng for the other Mab. Four of the five Mabs were stable during storage at 22 degrees C-23 degrees C for 24 h. These four Mabs are potentially useful for the immunodetection of Salmonella in foods and other samples.