Figure S1 shows BsAb 4 and BsAb 5 concentration over time in each mouse for half-life determination.

Figure S2 shows mesothelin expression on A431-Luc cells by flow cytometry.

Figure S3 shows that PBMCs and bispecific antibodies alone are insufficient to kill OVCAR8-Luc cancer cells.

Table S1 shows the amino acid sequences for the BsAbs investigated in the article.

Supplementary Method 1 explains the preparation of BsAb 5.

Mesothelin (MSLN) is a cell-surface protein that is expressed in many cancers, which makes it a popular target for Ab-based cancer therapy. However, MSLN is shed from cancer cells at high levels via proteases that cleave at its membrane-proximal C-terminal region. Shed MSLN accumulates in patients' fluids and tumors and can block Ab-based MSLN-targeting drugs from killing cancer cells. A previously established mAb, 15B6, binds MSLN at its protease-sensitive C-terminal region and does not bind shed MSLN. Moreover, 15B6 variable fragment (Fv)-derived chimeric antigen receptor T cells are not inhibited by shed MSLN and kill tumors in mice more effectively than mAb SS1 Fv-derived chimeric antigen receptor T cells, which bind an epitope retained in shed MSLN. In this study, we have established 15B6 Fv-derived MSLN × CD3 bispecific antibodies (BsAb) that target MSLN-expressing cancers. We identified our lead candidate BsAb 5 after screening multiple 15B6-derived BsAb formats in vitro for cytotoxic activity. BsAb 5 activates T cells to kill various cancer cell lines in a MSLN-specific manner. MSLN 296-591 His, a recombinant protein mimicking shed MSLN, does not inhibit 15B6-derived BsAb 5 but completely inhibits humanized SS1-derived BsAb 7. Furthermore, BsAb 5 inhibits and delays tumor growth and is not inhibited by MSLN 296-585 His in mice. Our findings indicate that by targeting the protease-sensitive region of MSLN, BsAb 5 has high MSLN-specific anticancer activity that is not inhibited by shed MSLN. BsAb 5 may be a promising immunotherapy candidate for MSLN-expressing cancers.

Biodistribution of LMB-249-PEG and SS1P

Mesothelin binding of PEGylated RITs

Fig S1. Description of DNA constructs Fig S2. Distribution of intra-tumoral injected trypan blue Fig S3. Combination of intra-tumoral immunotoxins with anti-CTLA-4 is well tolerated by mice. Fig S4. Anti-tumor effect of anti-CTLA-4 and LMB-100 depends on CD8+ cells. Fig S5. High dose of SS1P is needed for induction of complete remission. Fig S6A & B. Anti-tumor effect of an immunotoxin targeting human CD22 & (B) Combination of SS1P with anti-CTLA-4 lead to tumor regressionof 66C14 tumors not expressing MSLN. Fig S7. Intra-tumors injection of paclitaxel does not improve the anti-tumor activity of anti-CTLA-4. Table S1. Combination of RIT and anti-CTLA-4 induces long-term anti-tumor immunity.

ADP ribosylation activities of PEGylated RITs

Cell Killing Activities of control RIT

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

Flow cytometry analysis using 5 μg/ml of MSLN antibody showing that wild-type and TACE mutant cells express similar levels of surface MSLN.

Mouse weight after treatment with PEGylated RITs

Significance Mesothelin (MSLN) is a cell-surface protein that is a popular target for antibody-based therapies. We have identified shed MSLN as a major obstacle to successful antibody therapies and prepared a monoclonal antibody that inhibits shedding and makes very active CAR T cells whose activity is not blocked by shed MSLN and merits further preclinical development.

In the recent years, 3D metal printing technique is tremendously used for building products by vertical layer deposition for various engineering applications. In this work, semi-automatic movement setup was used to perform vertical layer deposition for different open and closed profiles utilizing gas metal arc welding (GMAW). The movement of welding torch along vertical direction is given manually, whereas the motion of the welding torch in the horizontal plane is completely autonomous. In order to achieve the precise controlled movement of the welding torch during metal printing on low carbon steel, electronic circuitry based on GRBL control was involved. Bead on bead trajectory was measured using coordinate measuring machine (CMM) to check the dimensional accuracy of the product. The micro-structural characterization using SEM image of the prepared samples is also done. Energy-Dispersive X-ray analytical technique was employed for elemental analysis or chemical characterization of the deposited specimens. The variations bead widths at a particular height for metal deposition of different profiles are measured. The micro-structural characterizations at the interface of two layers of metal deposition, also at the base metal and heat affected zone (HAZ) are performed for the observation of metallurgical changes in the structure. Maximum micro-hardness is observed at the HAZ and it gradually reduces towards the top layers in a particular pattern.

The human transferrin receptor (TFR) is overexpressed in most breast cancers, including preneoplastic ductal carcinoma in situ (DCIS). HB21(Fv)-PE40 is a single-chain immunotoxin (IT) engineered by fusing the variable region of a monoclonal antibody (HB21) against a TFR with a 40 kDa fragment of Pseudomonas exotoxin (PE). In humans, the administration of other TFR-targeted immunotoxins intrathecally led to inflammation and vascular leakage. We proposed that for treatment of DCIS, intraductal (i.duc) injection of HB21(Fv)-PE40 could avoid systemic toxicity while retaining its potent antitumor effects on visible and occult tumors in the entire ductal tree. Pharmacokinetic studies in mice showed that, in contrast to intravenous injection, IT was undetectable by enzyme-linked immunosorbent assay in blood following i.duc injection of up to 3.0 μg HB21(Fv)-PE40. We demonstrated the antitumor efficacy of HB21(Fv)-PE40 in two mammary-in-duct (MIND) models, MCF7 and SUM225, grown in NOD/SCID/gamma mice. Tumors were undetectable by In Vivo Imaging System (IVIS) imaging in intraductally treated mice within 1 wk of initiation of the regimen (IT once weekly/3 wk, 1.5 μg/teat). MCF7 tumor-bearing mice remained tumor free for up to 60 d of observation with i.duc IT, whereas the HB21 antibody alone or intraperitoneal IT treatment had minimal/no antitumor effects. These and similar findings in the SUM225 MIND model were substantiated by analysis of mammary gland whole mounts, histology, and immunohistochemistry for the proteins Ki67, CD31, CD71 (TFR), and Ku80. This study provides a strong preclinical foundation for conducting feasibility and safety trials in patients with stage 0 breast cancer.

A personal transporter vehicle, called segway, is based on the stabilization principle of inverted pendulum system. In this work, the bond graph model of segway with PI control is developed. The simulation results for the forward and backward motion of segway are presented as pitch angle, speed of the vehicle with respect to time. For the turning of the vehicle, controller based on Ackermann steering mechanism is adopted to modulate the voltages of two motors. The results of turning motion of the segway are also presented as yaw angle response of main vehicle body. The focus of this paper is to develop the dynamic model and simulate the response of segway using non-model-based control system design. The objective of the work is accomplished by developing the stabilization controller based on PI control scheme using bond graph approach. Also, the swing up controller is developed for nonlinear behaviour of the segway.

In construction industry, the conventional way of construction of buildings is a costly, time consuming and a labour-intensive job. Besides that, many on-site fatalities happen during the construction activity and the climate can delay construction activity. 3-D printer robot resolves all these problems. Also, it can be used for intricate building designs and for construction of buildings in remote locations or in epidemic situations. The objective of the project is to make an autonomous robot for 3-D printing a simple civil structure. Three lead screws will provide the movement in the vertical as well as in horizontal directions and a nozzle connected to the horizontal lead screw will be used for pouring the construction-material layer by layer. Thereafter, structural analysis using Finite Element Method (FEM) has been done on critical parts like lead screw and top plate. Bond graphs for buggy and overall lead screw system have also been used to analyze the response of the system. Furthermore, the wring diagram is also developed and presented in the paper. Future research is required for the development of actual robot for building multi-storied structures, on the design of nozzle and type of material used for pouring.

We investigated the role of mesothelin (Msln) and thymocyte differentiation antigen 1 (Thy1) in the activation of fibroblasts across multiple organs and demonstrated that Msln-/- mice are protected from cholestatic fibrosis caused by Mdr2 (multidrug resistance gene 2) deficiency, bleomycin-induced lung fibrosis, and UUO (unilateral urinary obstruction)-induced kidney fibrosis. On the contrary, Thy1-/- mice are more susceptible to fibrosis, suggesting that a Msln-Thy1 signaling complex is critical for tissue fibroblast activation. A similar mechanism was observed in human activated portal fibroblasts (aPFs). Targeting of human MSLN* aPFs with two anti-MSLN immunotoxins killed fibroblasts engineered to express human mesothelin and reduced collagen deposition in livers of bile duct ligation (BDL)- injured mice. We provide evidence that antimesothelin-based therapy may be a strategy for treatment of parenchymal organ fibrosis.