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Supplementary Data from GPC1-Targeted Immunotoxins Inhibit Pancreatic Tumor Growth in Mice via Depletion of Short-lived GPC1 and Downregulation of Wnt Signaling

Supplementary Figure 3. Graph showing growth of xenografted MKN28 gastric tumors following treatment with paclitaxel alone or in combination with RG7787 over a 60 day time course.

PDF file - 626K, Purified recombinant immunotoxins

Production and screening of ZZ-PE mutant fusion proteins

PDF - 261K, Supplementary methods related to the IHC protocol and supplementary figure legends.

Fig S1. H89 increased ADP-ribosylation and MCL-1 degradation in SEM cells Fig S2. Combination of RIT and H89 lowers MCL1 and induces PARP-cleavage Fig S3. PKA regulator, S6K1-, or ROCK2-inhibitor show little change in LMB-11 activity Fig S4. Combination of LMB-11 and S6K1 inhibitor PF4708671 enhances MCL1 degradation but not ADP-ribosylation Table S1. Cytotoxic activity (IC50) of immunotoxins with and without H89 Table S2. In vitro screening for H89 inhibited kinases

Supplemental Information. Includes Supplementary Methods section and legends for Supplemental Figures

Immune checkpoint blockade using antibodies to cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) benefits a limited number of cancer patients. SS1P and LMB-100 are immunotoxins that target mesothelin. We observed delayed responses to SS1P in patients with mesothelioma suggesting that antitumor immunity was induced. Our goal was to stimulate antitumor immunity by combining SS1P or LMB-100 with anti–CTLA-4. We constructed a BALB/c breast cancer cell line expressing human mesothelin (66C14-M), which was implanted in one or two locations. SS1P or LMB-100 was injected directly into established tumors and anti–CTLA-4 administered i.p. In mice with two tumors, one tumor was injected with immunotoxin and the other was not. The complete regression rate was 86% for the injected tumors and 53% for the uninjetced tumors. No complete regressions occurred when drugs were given separately. In regressing tumors, dying and dead tumor cells were intermingled with PMNs and surrounded by a collar of admixed eosinophils and mononuclear cells. Tumor regression was associated with increased numbers of tumor-infiltrating CD8+ cells and blocked by administration of antibodies to CD8. Surviving mice were protected from tumor rechallenge by 66C14 cells not expressing mesothelin, indicating the development of antitumor immunity. The antitumor effect was abolished when a mutant noncytotoxic variant was used instead of LMB-100, showing that the antitumor response is not mediated by recognition of a foreign bacterial protein. Our findings support developing a therapy composed of immunotoxins and checkpoint inhibitors for patients. Cancer Immunol Res; 5(8); 685–94. ©2017 AACR.

Supplementary Figure Legends - PDF file 88K, Supplementary Figure Legends 1-9

Purpose:LMB-100 is a recombinant immunotoxin (iTox) consisting of a mesothelin-binding Fab for targeting and a modified Pseudomonas exotoxin A payload. Preclinical studies showed that combining taxanes with iTox results in synergistic antitumor activity. The objectives of this phase I/II study were to determine the MTD of LMB-100 when administered with nanoalbumin bound (nab)-paclitaxel to patients with previously treated advanced pancreatic adenocarcinoma and to assess the objective response rate.Patients and Methods:Patients (n = 20) received fixed-dose nab-paclitaxel (125 mg/m2 on days 1 and 8) with LMB-100 (65 or 100 μg/kg on days 1, 3, and 5) in 21-day cycles for 1–3 cycles.Results:Fourteen patients were treated on the dose escalation and an additional six in the phase II expansion. MTD of 65 μg/kg was established for the combination. Dose-limiting toxicity resulting from capillary leak syndrome (CLS) was seen in two of five patients treated at 100 μg/kg and one of six evaluable phase I patients receiving the MTD. Severity of CLS was associated with increases in apoptotic circulating endothelial cells. LMB-100 exposure was unaffected by anti-LMB-100 antibody formation in five of 13 patients during cycle 2. Seven of 17 evaluable patients experienced >50% decrease in CA 19-9, including three with previous exposure to nab-paclitaxel. One patient developed an objective partial response. Patients with biomarker responses had higher tumor mesothelin expression.Conclusions:Although clinical activity was observed, the combination was not well tolerated and alternative drug combinations with LMB-100 will be pursued.

Semiquantitative assessment of mesothelin expression using H-score in mesothelioma patient derived xenografts.

Purpose: Many solid tumors express cell surface mesothelin making them attractive targets for antibody-based therapies of cancer. SS1P [antimesothelin(Fv)PE38] is a recombinant immunotoxin (RIT) that has potent cytotoxic activity on several cancer cell lines and clinical activity in mesothelioma patients. Pancreatic cancers express mesothelin and are known to be resistant to most chemotherapeutic agents. The goal of this study is to treat pancreatic cancer with RIT by targeting mesothelin.Experimental Design: We measured the cytotoxic activity of an antimesothelin immunotoxin on pancreatic cancer cells. We also measured the levels of several pro- and antiapoptotic proteins, as well as the ability of TNF-related apoptosis-inducing ligand (TRAIL) or the anti-TRAIL receptor 2 agonist antibody (HGS-ETR2) to kill pancreatic cells, and the cytotoxic activity of the two agents together in cell culture and against tumors in mice.Results: In two pancreatic cancer cell lines, immunotoxin treatment inhibited protein synthesis but did not produce significant cell death. The resistant lines had low levels of the proapoptotic protein Bak. Increasing Bak expression enhanced the sensitivity to immunotoxins, whereas Bak knockdown diminished it. We also found that combining immunotoxin with TRAIL or HGS-ETR2 caused synergistic cell death, and together triggered caspase-8 recruitment and activation, Bid cleavage and Bax activation. Combining SS1P with HGS-ETR2 also acted synergistically to decrease tumor burden in a mouse model.Conclusion: Our data show that low Bak can cause cancer cells to be resistant to immunotoxin treatment and that combining immunotoxin with TRAIL or a TRAIL agonist antibody can overcome resistance. Clin Cancer Res; 17(18); 5926–34. ©2011 AACR.

Dose adjustments.

Supplemental Figure 4

Figure S1. KOPN-8 from murine BM has similar levels of CD22 and RIT sensitivity as in cell culture Figure S2. LMB-11-Alexa 647 in KOPN-8 from murine BM reaches maximal signal intensity after only 1 h circulation time Figure S3. Compared to the most active rIT HA22, introduction of every new variable in LMB-11 reduced activity and increased exposure time against KOPN-8.

Conflict of Interest Form

Purpose: Amatuximab is a chimeric monoclonal antibody to mesothelin, a cell surface glycoprotein highly expressed in malignant pleural mesothelioma (MPM). On the basis of its synergy with chemotherapy in preclinical studies, we evaluated the antitumor activity of amatuximab plus pemetrexed and cisplatin in patients with unresectable MPM.Experimental Design: In a single-arm phase II study, amatuximab (5 mg/kg) was administered on days 1 and 8 with pemetrexed (500 mg/m2) and cisplatin (75 mg/m2) on day 1 of 21-day cycles for up to six cycles. Patients with response or stable disease received amatuximab maintenance until disease progression. Primary endpoint was progression-free survival (PFS) at 6 months. Secondary endpoints were overall survival (OS), response rate, and safety.Results: Eighty-nine patients were enrolled at 26 centers. Median of five cycles (range, 1–6) of combination treatment was administered, and 56 (63%) patients received amatuximab maintenance. Combination therapy resulted in no overlapping toxicities. Eleven patients (12.4%) had amatuximab-related hypersensitivity reactions. Responses included partial responses in 33 (40%) and stable disease in 42 (51%). Six-month PFS rate was 51% [95% confidence interval (CI), 39.1–62.3)], median PFS was 6.1 months (95% CI, 5.8–6.4), and median OS was 14.8 months (95% CI, 12.4–18.5) with 29 patients alive at data cut-off.Conclusions: Amatuximab with pemetrexed and cisplatin was well tolerated with objective tumor response or stable disease rate of 90% by independent radiologic review. Although PFS was not significantly different from historical controls, the median OS was 14.8 months with a third of patients alive and 5 continuing to receive amatuximab at the time of analysis. Clin Cancer Res; 20(23); 5927–36. ©2014 AACR.

Order of study arm enrollment is displayed graphically

Serious adverse events unrelated to research.

Recombinant immunotoxins (RIT) have been highly successful in cancer therapy due, in part, to the high cancer-specific expression of cell surface antigens such as mesothelin, which is overexpressed in mesothelioma, ovarian, lung, breast, and pancreatic cancers, but is limited in normal cells. RG7787 is a clinically optimized RIT consisting of a humanized anti-mesothelin Fab fused to domain III of Pseudomonas exotoxin A, in which immunogenic B-cell epitopes are silenced. To enhance the therapeutic efficacy of RITs, we conducted a kinome RNAi sensitization screen, which identified discoidin domain receptor 1 (DDR1), a collagen-activated tyrosine kinase, as a potential target. The collagen/DDR1 axis is implicated in tumor–stromal interactions and potentially affects tumor response to therapy. Therefore, we investigated the effects of DDR1 on RIT. Knockdown of DDR1 by siRNA or treatment with inhibitor, 7rh, greatly enhanced the cytotoxic activity of RG7787 in several cancer cell lines. Investigation into the mechanism of action showed DDR1 silencing was associated with decreased expression of several ribosomal proteins and enhanced inhibition of protein synthesis. Conversely, induction of DDR1 expression or collagen-stimulated DDR1 activity protected cancer cells from RG7787 killing. Moreover, the combination of RG7787 and DDR1 inhibitor caused greater shrinkage of tumor xenografts than either agent alone. These data demonstrate that DDR1 is a key modulator of RIT activity and represents a novel therapeutic strategy to improve targeting of mesothelin-expressing cancers. Cancer Res; 76(6); 1560–8. ©2015 AACR.

Adverse events attributed to nab-paclitaxel

Semiquantitative assessment of mesothelin expression using H-score in mesothelioma patient derived xenografts. (A) NCI-Meso16: tumor with areas of strong membrane staining and intermingling of negative areas (H-score 230). (B) NCI-Meso21: tumor shows only focal membrane positivity (H-score 50). (C) NCI-Meso29: tumor shows nearly uniform strong membrane positivity (H-score 290). Original magnification 400x.

Supplementary Methods Supplementary Reference Supplementary Figure Legends Supplementary Table Supplementary Figures Suppl Fig 1. Effect of DDR1 knockdown on internalization of alexa-647 labeled RG7787 Fig S2. Effect of DDR1 knockdown on mesothelin expression Fig S3. KB or A431/H9 cells were transfected with control siRNA or DDR1 siRNAs Fig S4. Effect of collagen I on DDR1 kinase activation in A) A431/H9 and B) KLM1 Fig S5. Nilotinib enhances cytotoxicity of RG7787 Fig S6. Combination of 7rh enhances cytotoxicity of native PE and HB21 Fig S7. 7rh inhibits DDR1 phosphorylation in A431/H9 tumors Table S1. siRNA and primer sequences used in this study

Purpose: Recombinant immunotoxins (rITs) targeting CD22 are highly active in hairy cell leukemia, but less so in acute lymphoblastic leukemia (ALL). This study aims to understand the variable activity of an rIT against ALL toward improving responses in clinical application.Experimental Design: We determined in vitro activity of rITs by WST-8 assays and the time needed to kill ALL cell lines and patient-derived ALL blasts by flow cytometry. The findings were translated into two systemic ALL xenograft models. Differences in time needed to kill KOPN-8 cells for distinct rITs were addressed biochemically.Results: In vitro activity (IC50) of anti-CD22 rIT varied 210-fold from 0.02 to 4.6 ng/mL. Activity also varied greatly depending on the time ALL cells were exposed to immunotoxin from < 30 minutes to > 4 days. For KOPN-8, the difference in exposure time was related to intracellular rIT processing. We showed in newly developed ALL xenograft models, where immunotoxins have a short half-life, that the needed exposure time in vitro predicted the responses in vivo. By replacing bolus dose with small doses at frequent intervals or with continuous infusion, responses were substantially improved. We confirmed exposure time variability on patient-derived ALL samples and showed a correlation between exposure time needed to reach maximal cytotoxicity in vitro and their clinical response.Conclusions: The exposure time needed for rITs targeting CD22 to kill ALL cells varies widely. Our results suggest that ALL patients would have a better response rate to anti-CD22 immunotoxins if treated by continuous infusion rather than by bolus injections. Clin Cancer Res; 22(19); 4913–22. ©2016 AACR.

Supplementary Methods - PDF file 94K, The supplementary methods section describes the following: Construction, expression and purification of different EGFRvIII deletion mutants, cloning of variable heavy (VH) and variable light (VL) domains of P588 and D2C7 mAb, surface plasmon resonance, stability assay, and immunohistochemistry of frozen D270MG brain tumor tissue

Supplementary Figures 1-9 - Supplementary Figures 1-9 - PDF file 516K, Supplementary Figure S1. Deduced amino acid sequence of the D2C7 scFv cloned from D2C7 hybridoma. VH (A) and VL (B) antigen-binding regions of the D2C7 scFv. Amino acid numbering and CDR (underlined) delimitation were determined according to the Kabat database. Supplementary Figure S2. Purified D2C7-(scdsFv)-PE38KDEL immunotoxin. For this analysis, 200 ng of the purified D2C7-(scdsFv)-PE38KDEL immunotoxin was run on NuPAGE 4-12% Bis-Tris gel (Invitrogen), along with a standard molecular weight protein marker. Supplementary Figure S3. Surface plasmon resonance analysis of D2C7-(scdsFv)-PE38KDEL. Binding kinetics and affinity constants of D2C7-(scdsFv)-PE38KDEL for EGFRwt (A) and EGFRvIII (B) were determined by surface plasmon resonance against bacterially expressed recombinant EGFRwt or EGFRvIII extracellular domain (ECD) proteins. The association and dissociation rates from the sensogram were KA = 6.3 �~ 108 M-1 and KD = 1.6 �~ 10-9 M against EGFRwt and KA = 7.8 �~ 108 M-1 and KD = 1.3 �~ 10-9M against EGFRvIII. Supplementary Figure S4. Flow cytometric analysis of CD133 and D2C7 mAb on 43 and D2159MG glioblastoma xenograft cells. FACS analysis demonstrates the double staining of CD133 and D2C7 (percentage of cells in Q2) on 71% (A) and 86% (B) of cells freshly isolated from glioblastoma xenografts 43 and D2159MG, respectively. Q1 represents the percentage of cells positive for D2C7 only; Q4 represents the percentage of cells positive for CD133 only and Q3 represents the percentage of cells negative for both CD133 and D2C7. Supplementary Figure S5. Stability of D2C7-(scdsFv)-PE38KDEL. D2C7-(scdsFv)-PE38KDEL was incubated with 0.2% BSA/PBS at 40 ��g/mL (A) and 7.5 ��g/mL (B), over a 7 day period at 37{degree sign}C and then assayed for cytotoxic activity on NR6M cells. Cytotoxicity data are given as IC50 value versus radioactive leucine incorporation. Supplementary Figure S6. Flow cytometric analysis of D2C7 mAb and mouse EGFR antibody on normal mousekidney fibroblast cells (499) or avian sarcoma virus transformed mouse glioma cells (539). FACS analysis demonstrates the reactivity of mouse EGFR (msEGFR) antibody (pink peaks) to EGFR expressing 499 (A) and 539 (C) cells and the absence of D2C7 (pink peaks) reactivity to both 499 (B) and 539 (D) cells, confirming that D2C7 is human specific and does not react with murine EGFR. Isotype-specific antibody (IgG1) was used as control (filled purple peaks). Supplementary Figure S7. Survival of mice injected intracranially with 43 xenograft cells (A), NR6M cells (B), or D270MG xenograft cells (C). Mice were injected intracranially with 1x105 43, NR6M, or D270MG cells and were checked daily for survival. Data are presented as the survival time in days versus percentage of mice surviving. Supplementary Figure S8. Toxicity of D2C7-(scdsFv)-PE38KDEL immunotoxin administered to NSG mice. Different doses of D2C7-(scdsFv)-PE38KDEL were delivered intracranially over a 7 day period to NSG mice (5 mice/group). Animals were monitored for toxicity related death. Data are expressed as percentage of mice surviving versus time. Supplementary Figure S9. Immunohistochemical detection of D2C7-(scdsFv)-PE38KDEL distribution in D270MG orthotopic model after immunotoxin treatment. Acetone-fixed frozen D270MG-sham (negative control) (a) and D270MG-D2C7-(scdsFv)-PE38KDEL group (test) (c) sections were stained with 1.0 ��g of mouse-anti-PE38KDEL antibodies. Tumor sections from D270MG-D2C7-(scdsFv)-PE38KDEL group, pre-stained with D2C7-(scdsFv)-PE38KDEL was used as positive control (b)

Purpose: The EGF receptor gene (EGFR) is most frequently amplified and overexpressed, along with its deletion mutant, EGFRvIII, in glioblastoma. We tested the preclinical efficacy of the recombinant immunotoxin, D2C7-(scdsFv)-PE38KDEL, which is reactive with a 55-amino acid (AA) region present in the extracellular domain of both EGFRwt (583-637 AAs) and EGFRvIII (292-346 AAs) proteins.Experimental Design: The binding affinity and specificity of D2C7-(scdsFv)-PE38KDEL for EGFRwt and EGFRvIII were measured by surface-plasmon resonance and flow cytometry. In vitro cytotoxicity of D2C7-(scdsFv)-PE38KDEL was measured by inhibition of protein synthesis in human EGFRwt-transfected NR6 (NR6W), human EGFRvIII-transfected NR6 (NR6M), EGFRwt-overexpressing A431-epidermoid-carcinoma, and glioblastoma xenograft cells (43, D08-0493MG, D2159MG, and D270MG). In vivo antitumor efficacy of D2C7-(scdsFv)-PE38KDEL was evaluated using 43, NR6M, and D270MG orthotopic tumor models.Results: The KD of D2C7-(scdsFv)-PE38KDEL for EGFRwt and EGFRvIII was 1.6 × 10−9 mol/L and 1.3 × 10−9 mol/L, respectively. Flow cytometry with NR6W and NR6M cells confirmed the specificity of D2C7-(scdsFv)-PE38KDEL for EGFRwt and EGFRvIII. The D2C7-(scdsFv)-PE38KDEL IC50 was 0.18 to 2.5 ng/mL on cells expressing EGFRwt (NR6W, A431, 43, and D08-0493MG). The D2C7-(scdsFv)-PE38KDEL IC50 was approximately 0.25 ng/mL on EGFRvIII-expressing cells (NR6M) and on EGFRwt- and EGFRvIII-expressing glioblastoma xenograft cells (D2159MG and D270MG). Significantly, in intracranial tumor models of 43, NR6M, and D270MG, treatment with D2C7-(scdsFv)-PE38KDEL by convection-enhanced delivery prolonged survival by 310% (P = 0.006), 28% (P = 0.002), and 166% (P = 0.001), respectively.Conclusions: In preclinical studies, the D2C7-(scdsFv)-PE38KDEL immunotoxin exhibited significant potential for treating brain tumors expressing EGFRwt, EGFRvIII, or both. Clin Cancer Res; 19(17); 4717–27. ©2013 AACR.

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New gene expressed in prostate (NGEP) is a prostate-specific polytopic membrane protein found at high concentrations at cell:cell contact regions. To determine if NGEP is a useful target for antibody-based therapy of prostate cancer, we performed an immunohistochemical analysis of 126 human prostate carcinoma samples using polyclonal anti-NGEP sera and found that 91% of the cancers express NGEP protein. To elucidate the topology of NGEP and guide the development of monoclonal antibodies (mAb) reacting with the extracellular regions of NGEP, a hemagglutinin epitope tag was inserted at several positions within the NGEP sequence. The tagged proteins were expressed in 293T cells and locations of the tags were determined by immunofluorescence in intact or permeabilized cells. The results indicate that NGEP contains eight transmembrane domains with both the NH2 and COOH termini of NGEP located inside the cell. We produced mAb to three regions that are predicted to be intracellular based on the epitope tag data (amino acids 1-352, 441-501, and 868-933), and as predicted, the mAb only detected the protein in permeabilized cells. NGEP is a glycoprotein with predicted glycosylation sites at N809 and N824. When these residues were converted to glutamine, glycosylation was abolished, confirming that the residues are extracellular. Our findings on the expression and the orientation of the NGEP protein serve as an important framework for the development of mAb targeting the extracellular regions of NGEP that could be used for prostate cancer immunotherapy. [Cancer Res 2008;68(15):6306–12]

PDF file - 93K, Summary of the results of SS1P toxicity after IR knock down or addition of inhibitors in KB31 cells.

PDF file - 125K

PDF file - 343K, A schematic of RIT intracellular trafficking pathways

PDF file - 697K, siRNA siIR-�2, which does not knock down IR expression, did not affect SS1P toxicity (S1); Treatment by IT reduced IR protein levels (S2); LRP1B is expressed in KB31 cells (S3); SS1P internalization was not enhanced in KB31 cells after IR knock down (S4); Inhibitors of the IR signal pathway did not affect SS1P induced toxicity (S5); IR-A and IR-B isoform expression in cell lines (S6).

PDF file - 39K, Position profile of the ECS concentration of complexed RIT at different times

Supplementary Figure 2 from Eradication of Tumor Colonization and Invasion by a B Cell–Specific Immunotoxin in a Murine Model for Human Primary Intraocular Lymphoma

Human primary intraocular lymphoma (PIOL) is predominantly a B cell–originated malignant disease with no appropriate animal models and effective therapies available. This study aimed to establish a mouse model to closely mimic human B-cell PIOL and to test the therapeutic potential of a recently developed immunotoxin targeting human B-cell lymphomas. Human B-cell lymphoma cells were intravitreally injected into severe combined immunodeficient mice. The resemblance of this tumor model to human PIOL was examined by fundoscopy, histopathology, immunohistochemistry, and evaluated for molecular markers. The therapeutic effectiveness of immunotoxin HA22 was tested by injecting the drug intravitreally. Results showed that the murine model resembles human PIOL closely. Pathologic examination revealed that the tumor cells initially colonized on the retinal surface, followed by infiltrating through the retinal layers, expanding preferentially in the subretinal space, and eventually penetrating through the retinal pigment epithelium into the choroid. Several putative molecular markers for human PIOL were expressed in vivo in this model. Tumor metastasis into the central nervous system was also observed. A single intravitreal injection of immunotoxin HA22 after the establishment of the PIOL resulted in complete regression of the tumor. This is the first report of a murine model that closely mimics human B-cell PIOL. This model may be a valuable tool in understanding the molecular pathogenesis of human PIOL and for the evaluation of new therapeutic approaches. The results of B cell–specific immunotoxin therapy may have clinical implications in treating human PIOL. (Cancer Res 2006; 66(21): 10586-93)

PDF file - 100K, This table gives all the parameters and their values used in the model

Supplementary Figure 1 from Eradication of Tumor Colonization and Invasion by a B Cell–Specific Immunotoxin in a Murine Model for Human Primary Intraocular Lymphoma

Supplementary Figure 1, Tables 1-4 from Topology of NGEP, a Prostate-Specific Cell:Cell Junction Protein Widely Expressed in Many Cancers of Different Grade Level

PDF 885K

Supplementary Table 1 from Differential Cellular Internalization of Anti-CD19 and -CD22 Immunotoxins Results in Different Cytotoxic Activity

PDF file - 208K, Contains all equations of the model and their derivations

PDF file - 66K, Kinetic steps that control the surface receptor concentration