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Abbreviated Title: LMB-100 in Mesothelioma NIH Protocol \#: 16C0127 NCT \#: NCT02798536 Version Date: February 16, 2021 Title: A Phase I Study of the Mesothelin-Targeted Immunotoxin LMB-100 with or without NabPaclitaxel (Abraxane) in Patients with Malignant Mesothelioma  
| NCI Principal Investigator:   | Raffit Hassan, M.D. Thoracic and GI Malignancies Branch (TGMB), CCR, NCI Building 10-CRC, Room 4-5330 9000 Rockville Pike Bethesda, MD 20892 Phone: 240-760-6232 E-mail: hassanr@mail.nih.gov   |
|-------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| Drug Name:    | LMB-100 (formerly RO6927005)         |
|---------------|--------------------------------------|
| IND Number:   | 152907                               |
| Sponsor:      | Center for Cancer Research, NCI, NIH |
| Manufacturer: | F. Hoffmann-La Roche                 |  
Commercial Agent Nab-paclitaxel
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precis background

Although mesothelioma patients with a limited tumor burden may benefit from surgical resection, most patients have advanced disease at diagnosis and are not candidates for cytoreductive surgery.  
For mesothelioma patients who are not eligible for curative surgery, the median survival with supportive care alone is 6 months whereas with the current standard treatment, a combination of cisplatin and pemetrexed, the median survival is 12 months.  
Mesothelin, a tumor differentiation antigen, is expressed in over 95% of epithelioid mesothelioma. Mesothelin is a suitable candidate for targeted therapy due to its very limited expression in normal human tissue and its high expression in several tumors including mesothelioma.  
LMB-100 is a novel recombinant anti-mesothelin immunotoxin developed for the treatment of patients with solid tumors that express mesothelin. Mesothelin is targeted by linking a humanized fragment of the anti-mesothelin Fab to a de-immunized Pseudomonas exotoxin (PE)
The clinical use of first generation immunotoxins such as SS1P was hampered mainly by their high immunogenicity. LMB-100 is a next generation PE-fusion protein that has been protein-engineered to maximally reduce its immunogenicity. LMB-100 has shown broad activity against different mesothelin-expressing cancer cell lines and tumor xenograft models.  
The combination of LMB-100 plus nab-paclitaxel has shown anti-tumor efficacy in different mesothelioma patient derived xenograft (PDX) models compared to LMB-100 or nab-paclitaxel alone.
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objectives

To identify the recommended phase 2 dose (RP2D) of LMB-100 +/- nab-paclitaxel in patients with treatment refractory advanced epithelioid or biphasic mesothelioma and evaluate potential efficacy of the identified RP2D.
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eligibility

Age ≥ 18 years Histologically confirmed advanced pleural or peritoneal mesothelioma Subjects must have had at least 1 prior chemotherapy regimen with last dose of previous therapy occurring at least 3 weeks before the start of study treatment Subjects enrolling in Arms B1 and B2 must not have received paclitaxel or nab-paclitaxel within 4 months prior to initiation of study therapy Adequate organ function Participants with CNS metastases or prior pneumonectomy are excluded
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design

This is a Phase I, open-label study to evaluate the safety, pharmacokinetics, and activity of LMB-100 +/- nab-paclitaxel in patients with treatment refractory advanced epithelioid or biphasic mesothelioma.  
Subjects in Arms A1 and A2 (completed) will receive LMB-100 monotherapy; subjects in Arms B1 and B2 will receive LMB-100+nab-paclitaxel LMB-100 will be administered intravenously on days 1, 3 and 5 of each 21 day cycle for up to 4 cycles in Arms A1 and A2 and up to 2 cycles in Arms B1 and B2 Nab-paclitaxel (Arms B1 and B2 only) will be administered intravenously on days 1 and 8 of each 21 day cycle for up to 6 cycles Tumor response will be assessed after every 2 cycles Optional tumor biopsies may be collected at baseline and after 2 cycles of therapy The accrual ceiling will be set at 34
3  
| TABLE OF CONTENTS                                                                                                                              |                                                                                                                                                   |                                                                                                                                     |
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| PRÉCIS .                                                                                                                                       |                                                                                                                                                   |                                                                                                                                     |
| TABLE OF CONTENTS                                                                                                                              |                                                                                                                                                   |                                                                                                                                     |
| STATEMENT OF COMPLIANCE                                                                                                                        |                                                                                                                                                   |                                                                                                                                     |
| Introduction                                                                                                                                   |                                                                                                                                                   |                                                                                                                                     |
| l                                                                                                                                              | Study Objectives .                                                                                                                                |                                                                                                                                     |
| 1.1                                                                                                                                            | 1.1.1                                                                                                                                             | Primary Objective: ...............................................................................................................9 |
| 1.1.2                                                                                                                                          | Secondary Objectives                                                                                                                              |                                                                                                                                     |
| Exploratory Objectives: .......................................................................................................9               |                                                                                                                                                   |                                                                                                                                     |
| 1.1.3                                                                                                                                          |                                                                                                                                                   |                                                                                                                                     |
| 1.2                                                                                                                                            | Background and Rationale ........................................................................................................10               |                                                                                                                                     |
| 1.2.1                                                                                                                                          | Background on Mesothelioma ...........................................................................................10                          |                                                                                                                                     |
| 1.2.2                                                                                                                                          | Mesothelin as a target for cancer therapy ....................................................................10                                  |                                                                                                                                     |
| LMB-100.....................................................................................................................................12 |                                                                                                                                                   |                                                                                                                                     |
| 1.2.3                                                                                                                                          |                                                                                                                                                   |                                                                                                                                     |
| 1.2.4                                                                                                                                          | Comparison of toxicity and immunogenicity of LMB-100 to SS1P ...................26                                                                |                                                                                                                                     |
| 1.2.5                                                                                                                                          | Rationale for the study .........................................................................................................27               |                                                                                                                                     |
| 2                                                                                                                                              | Eligibility Assessment and Enrollment.....................................................................................28                      |                                                                                                                                     |
| 2.1                                                                                                                                            | Eligibility Criteria ..........................................................................................................................28 |                                                                                                                                     |
| 2.1.1                                                                                                                                          | Inclusion Criteria (All Arms)............................................................................................28                       |                                                                                                                                     |
| 2.1.2                                                                                                                                          | Exclusion Criteria (All Arms)                                                                                                                     |                                                                                                                                     |
| 2.1.3                                                                                                                                          | Exclusion Criteria (Arm B only)                                                                                                                   |                                                                                                                                     |
| 2.2                                                                                                                                            | Screening Evaluation ..........................................................................................................30                 |                                                                                                                                     |
| 2.3                                                                                                                                            | Registration Procedures ...............................................................................................................31         |                                                                                                                                     |
| Treatment Assignment Procedures .............................................................................31                                |                                                                                                                                                   |                                                                                                                                     |
| 2.3.1                                                                                                                                          |                                                                                                                                                   |                                                                                                                                     |
| 3                                                                                                                                              | Study Implementation .......................................................................................................................32    |                                                                                                                                     |
| 3.1                                                                                                                                            | Study Design ............................................................................................................................32       |                                                                                                                                     |
| 3.1.1                                                                                                                                          | Dose Limiting Toxicity (Dose de-escalation cohorts only) ......................................33                                                 |                                                                                                                                     |
| 3.1.2                                                                                                                                          | Dose De-Escalation (Arm A1)                                                                                                                       |                                                                                                                                     |
| 3.1.3                                                                                                                                          | ,Dose De-Escalation (Arm B1)                                                                                                                      |                                                                                                                                     |
| 3.2                                                                                                                                            | D Drug Administration ...................................................................................................................36       |                                                                                                                                     |
| 3.2.1                                                                                                                                          | LMB-100.............................................................................................................................36            |                                                                                                                                     |
| Nab- paclitaxel (Arms B1 and B2 Only) ..................................................................39                                     |                                                                                                                                                   |                                                                                                                                     |
| 3.2.2                                                                                                                                          |                                                                                                                                                   |                                                                                                                                     |
| 3.2.3                                                                                                                                          | Co-administration of Nab-paclitaxel and LMB-100 (Arms B1 and B2 only)....40                                                                       |                                                                                                                                     |
| 3.3                                                                                                                                            | Dose Modifications ............................................................................................................40                 |                                                                                                                                     |  
| 3.3.1   | For Arm A1 Subjects Treated at 170 mcg/kg only 40                                                          |
|---------|------------------------------------------------------------------------------------------------------------|
| 3.3.2   | For all Subjects 40                                                                                        |
| 3.3.3   | Nab-paclitaxel 42                                                                                          |
| 3.4     | Study Calendar 43                                                                                          |
| 3.4.1   | Arms A1 and A2 Calendar 43                                                                                 |
| 3.4.2   | Arms B1 and B2 Calendar 46                                                                                 |
| 3.5     | Criteria for Removal from Protocol Therapy and Off Study Criteria 49                                       |
| 3.5.1   | Criteria for removal from protocol therapy 49                                                              |
| 3.5.2   | Follow Up 49                                                                                               |
| 3.5.3   | Off-Study Criteria 49                                                                                      |
| 3.5.4   | Off Protocol Therapy and Off Study Procedure 49                                                            |
| 4       | Concomitant Medications/Measures 49 4.1.1 Permitted Therapy 49 4.1.2 Prohibited Therapy 50                 |
| 5       | Biospecimen Collection 50                                                                                  |
| 5.1     | Correlative Studies for Research/Pharmacokinetic Studies 50                                                |
| 5.1.1   | Pharmacokinetic Assessments 50                                                                             |
| 5.1.2   | Assessment of anti-drug antibodies (ADAs) 51                                                               |
| 5.1.3   | Mesothelin and Megakaryocyte Potentiating Factor (MPF) Serum Samples: 52                                   |
| 5.1.4   | Retrospective Analysis of Mesothelin Expression in tumor tissue 53                                         |
| 5.1.5   | Cytokines for identification of mechanism for PE-mediated capillary leak syndrome 53                       |
| 5.1.6   | Gene expression-based characterization of the immune landscape before and after  treatment with LMB-100 54 |
| 5.1.7   | Mapping T cell epitopes in LMB-100 55                                                                      |
| 5.1.8   | Assessment of Myeloid Derived Suppressor Cells (MDSC) in Subjects with  Mesothelioma 56                    |
| 5.2     | Sample Collection schedule 57                                                                              |
| 5.2.1   | Arms A1 and A2 sample collection schedule 57                                                               |
| 5.2.2   | Arms B1 and B2 Sample Collection Schedule 60                                                               |
| 5.3     | Sample Storage, Tracking and Disposition 63                                                                |
| 5.3.1   | Clinical Pharmacology Program 63                                                                           |
| 5.3.2   | Leidos Biomedical, Inc. Lab 63                                                                             |  
Version Date: 02/16/2021  
| Version Date: 02/16/2021                                                                                                                              |                                                                                                                                                          |
|-------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------|
| 5.3.3                                                                                                                                                 | Laboratory of Dr. Raffit Hassan ................................................................................64                                       |
| NIH Flow Cytometry Laboratory ...........................................................................64                                           |                                                                                                                                                          |
| 5.3.4                                                                                                                                                 | Protocol Completion/Sample Destruction ..............................................................64                                                  |
| 5.3.5                                                                                                                                                 |                                                                                                                                                          |
| 6                                                                                                                                                     | DATA COLLECTION AND EVALUATION ....................................................................................................65                    |
| DATA COLLECTION                                                                                                                                       |                                                                                                                                                          |
| 6.1                                                                                                                                                   |                                                                                                                                                          |
| 6.2                                                                                                                                                   | DATA SHARING Plans ..............................................................................................................................65      |
| 6.2.1                                                                                                                                                 | Human Data Sharing Plan............................................................................................65                                    |
| 6.2.2                                                                                                                                                 | Genomic Data Sharing Plan...............................................................................................66                               |
| Response Criteria .................................................................................................................................66 |                                                                                                                                                          |
| 6.3                                                                                                                                                   |                                                                                                                                                          |
| 6.3.1                                                                                                                                                 | Definitions                                                                                                                                              |
| 6.3.2                                                                                                                                                 | Peritoneal Mesothelioma ..................................................................................................67                             |
| Pleural Mesothelioma .........................................................................................................72                      |                                                                                                                                                          |
| 6.3.3                                                                                                                                                 | Duration of Response                                                                                                                                     |
| 6.3.4                                                                                                                                                 |                                                                                                                                                          |
| 6.3.5                                                                                                                                                 | Progression-Free Survival                                                                                                                                |
| Objective Response Rate .....................................................................................................74                       |                                                                                                                                                          |
| 6.3.6                                                                                                                                                 |                                                                                                                                                          |
| 6.3.7                                                                                                                                                 | Disease Control Rate                                                                                                                                     |
| 6.3.8                                                                                                                                                 | OVERALL SURVIVAL                                                                                                                                         |
| 6.4                                                                                                                                                   | Toxicity Criteria ....................................................................................................................................74 |
| 7                                                                                                                                                     | NIH Reporting Requirements/Data and Safety Monitoring Plan ........................................74                                                    |
| 7.1                                                                                                                                                   | Definitions                                                                                                                                              |
| 7.2                                                                                                                                                   | OHSRP Office of Compliance and Training /IRB Reporting ..........................................74                                                      |
| 7.2.1                                                                                                                                                 | Expedited Reporting ................................................................................................................74                   |
| 7.2.2                                                                                                                                                 | IRB Requirements for PI Reporting at Continuing Review .....................................74                                                           |
| 7.3                                                                                                                                                   | NCI Clinical Director Reporting ..................................................................................................74                     |
| 7.4                                                                                                                                                   | NIH Required Data and Safety Monitoring Plan ....................................................................75                                      |
| 7.4.1                                                                                                                                                 | Principal Investigator/Research Team ...............................................................................75                                   |
| Safety Monitoring Committee (SMC) ....................................................................................75                              |                                                                                                                                                          |
| 7.4.2                                                                                                                                                 |                                                                                                                                                          |
| 8                                                                                                                                                     | Sponsor Safety Reporting ..................................................................................................................75            |
| 8.1                                                                                                                                                   | Definitions                                                                                                                                              |
| Adverse Event ....................................................................................................................................75  |                                                                                                                                                          |
| 8.1.1                                                                                                                                                 |                                                                                                                                                          |
| 8.1.2                                                                                                                                                 | Serious Adverse Event (SAE)                                                                                                                              |
| 8.1.3                                                                                                                                                 | Life-threatening                                                                                                                                         |
| Severity .                                                                                                                                            |                                                                                                                                                          |
| 8.1.4                                                                                                                                                 |                                                                                                                                                          |  
| Version Date: 02/16/2021 8.1.5 Relationship to Study Product 76 8.2 Assessment of Safety Events 76 8.3 Reporting of Serious Adverse Events 77 8.4 Reporting Pregnancy 77 8.4.1 Maternal exposure 77 8.4.2 Paternal exposure 78 8.5 Regulatory Reporting for Studies Conducted Under CCR-Sponsored IND 78 9 Clinical Monitoring Plan 78 10 Statistical Considerations 78 10.1 Arms A1 and A2 79 10.2 Arms B1 and B2 80 10.3 Justification of accrual ceiling 80 11 Collaborative Agreements 80 11.1 Clinical Trial Agreement 80 12 Human Subjects Protections 80 12.1 Rationale for Subject Selection 80 12.2 Participation of Children 81 12.3 Participation of Subjects Unable to Give Consent 81 12.4 Evaluation of Benefits and Risks/Discomforts 81 12.4.1 Risks from Study Drugs 81 12.4.2 Radiation Risks 81 12.5 Risks/Benefits Analysis 81 12.6 Consent Process and Documentation 82 12.6.1 Telephone re-consent procedure 82 13 REGULATORY AND OPERATIONAL CONSIDERATIONS 82 13.1 Quality Assurance and Quality Control 82 13.2 Conflict of Interest Policy 83 13.3 Confidentiality and Privacy 83 14 Pharmaceutical Information 84 14.1 LMB-100 (IND # 152907) 84 14.1.1 Source 84 14.1.2 Toxicity 84 14.1.3 Formulation and preparation 867   |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
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| 14.1.4   | Stability and Storage 86                                                          |
|----------|-----------------------------------------------------------------------------------|
| 14.1.5   | Administration procedures 86                                                      |
| 14.1.6   | Incompatibilities 86                                                              |
| 14.2     | Nab-paclitaxel 87                                                                 |
| 14.2.1   | Source 87                                                                         |
| 14.2.2   | Toxicity 87                                                                       |
| 14.2.3   | Formulation and preparation 88                                                    |
| 14.2.4   | Stability and Storage 89                                                          |
| 14.2.5   | Administration procedures 90                                                      |
| 14.2.6   | Incompatibilities 90                                                              |
| 15       | REFERENCES 91                                                                     |
| 16       | APPENDICES 95                                                                     |
| 16.1     | Appendix A - Performance Status Criteria 95                                       |
| 16.2     | Appendix B - Cockcroft-Gault Formula for Calculation of Creatinine Clearance ..96 |
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statement of compliance

The trial will be carried out in accordance with International Conference on Harmonisation Good Clinical Practice (ICH GCP) and the following:  
United States (US) Code of Federal Regulations (CFR) applicable to clinical studies (45 CFR Part 46, 21 CFR Part 50, 21 CFR Part 56, 21 CFR Part 312, and/or 21 CFR Part 812)
National Institutes of Health (NIH)-funded investigators and clinical trial site staff who are responsible for the conduct, management, or oversight of NIH-funded clinical trials have completed Human Subjects Protection and ICH GCP Training. The protocol, informed consent form(s), recruitment materials, and all participant materials will be submitted to the Institutional Review Board (IRB) for review and approval. Approval of both the protocol and the consent form must be obtained before any participant is enrolled. Any amendment to the protocol will require review and approval by the IRB before the changes are implemented to the study. In addition, all changes to the consent form will be IRB-approved; an IRB determination will be made regarding whether a new consent needs to be obtained from participants who provided consent, using a previously approved consent form.
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1 introduction

1.1 STUDY OBJECTIVES 1.1.1 Primary Objective:
To identify the recommended phase 2 dose (RP2D) of LMB-100 +/- nab-paclitaxel in patients with treatment refractory advanced epithelioid or biphasic mesothelioma.  
1.1.2 Secondary Objectives To define the pharmacokinetics characteristics of LMB-100 +/- nab-paclitaxel, including the number of effective cycles of LMB-100 therapy that can be given before the development of anti-LMB-100 antibodies To assess the safety and tolerability of LMB-100 +/- nab-paclitaxel in patients with treatment refractory epithelioid or biphasic mesothelioma To determine the duration of response, progression free survival and overall survival To determine the efficacy at the RP2D of LMB-100 +/- *nab*-paclitaxel with respect to objective response rate in patients with treatment refractory advanced epithelioid or biphasic mesothelioma.  
1.1.3 Exploratory Objectives:
To establish the correlation of response to treatment with tumor mesothelin expression; To evaluate changes in the tumor microenvironment following treatment with LMB100 +/- nab-paclitaxel To identify the mechanism for Pseudomonas exotoxin-mediated capillary leak syndrome To investigate the potential of soluble mesothelin levels and levels of megakaryocyte
potentiating factor to predict any therapeutic response  
1.2 BACKGROUND AND RATIONALE
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1.2.1 background on mesothelioma

Mesothelioma is a neoplasm originating from the mesothelial cells lining human body cavities. Mesothelioma may involve the pleura and less frequently, the peritoneum. Approximately 3000 new cases are diagnosed every year in the US alone. The epithelioid variant is the most common, comprising about 60 percent of all mesotheliomas. Malignant pleural mesothelioma is an aggressive disease with poor prognosis. Although patients with a limited tumor burden may benefit from surgical resection, most patients have advanced disease at diagnosis and are not candidates for cytoreductive surgery.[1] For patients who are not eligible for curative surgery, the median survival with supportive care alone is 6 months whereas with the current standard treatment, a combination of cisplatin and pemetrexed, the median survival is 12 months.[2] Peritoneal mesothelioma represents about one-fifth to one-third of all forms of mesothelioma; there are approximately 400 new cases in the United States each year.[3] Cytoreductive surgery and hyperthermic perioperative chemotherapy is the accepted initial management for suitable patients with peritoneal mesothelioma.[4-7] Peritoneal mesothelioma patients with surgically unresectable disease or whose medical co-morbidities preclude surgery are considered for palliative systemic therapy. Due to its relatively low incidence and inherent difficulties of radiologic assessment, few studies of systemic therapy have been conducted. Treatment recommendations are often extrapolated from pleural mesothelioma and outcomes are poor.
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1.2.2 mesothelin as a target

Mesothelin is a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein, which is present in a restricted set of normal adult tissues, such as the mesothelial lining of the pleura and pericardium. Immunohistochemistry has shown that mesothelin is highly expressed in nearly all epithelioid mesotheliomas as well as epithelial components of biphasic mesothelioma in addition to pancreatic ductal adenocarcinomas and in a high percentage of epithelial ovarian cancers and non-small cell lung cancer (NSCLC).[8] Although the normal biological function of mesothelin is unknown, growing evidence suggests that it may play a role in tumorigenesis and metastasis. Its limited expression in normal human tissue and high expression in tumor makes mesothelin an excellent target antigen for antibody-based immunotherapy.[9] Because of the high expression of mesothelin in many malignancies, a variety of agents are being developed to target mesothelin. Results of several ongoing clinical trials of immunotherapy agents directed against mesothelin have shown that targeting mesothelin is safe and does not result in toxicity to essential normal tissues. Both antibody-based therapies, as well as mesothelin vaccines, are being investigated.[10] The Laboratory of Molecular Biology (LMB) and the Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute have pioneered the use of mesothelin- targeted agents and clinical trials over the last decade.
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1.2.2.1 recombinant immunotoxins (rits) 1.2.2.1.1

RITs are antibody-based therapeutics that carry a toxin payload. RITs that target mesothelin contain a genetically engineered variant of Pseudomonas exotoxin A (PE) in which the native cellbinding domain of PE is replaced by a mesothelin-binding antibody fragment. In this way, the RIT binds specifically to mesothelin on the cell surface and gets internalized through endocytic processes. In the cytosol, PE catalyzes an irreversible, inactivating modification of eukaryotic Elongation Factor-2 (eEF-2). This prevents the elongation step of protein synthesis, halting production of new cellular proteins, a stressor that triggers apoptosis. This mechanism of action results in cytotoxicity to both proliferating and non-dividing cells, therefore proper targeting is critical to the safety profile.[11] Tumor-targeted bacterial toxins that inhibit the protein synthesis of cancer cells have previously shown signs of preclinical and clinical efficacy. For instance, SS1P, a classical immunotoxin format molecule consisting of the mouse anti-mesothelin Fv, SS1 and a 38 kDa PE toxin fragment (PE38) is one example. Besides SS1P, the most clinically advanced PE-based immunotoxins are moxetumomab pasudotox, a CD22-targeted fusion protein in development by LMB/Astra Zeneca/
Medimmune, and LMB2, a CD25-targeted PE fusion protein developed by LMB. Both agents have shown encouraging signs of efficacy in different hematological malignancies and are currently in Phase II and III clinical trials. Denileukin difitox is the only immunotoxin approved by the US Food and Drug Administration (FDA); a fusion protein of diphtheria toxin with interleukin-2 that is clinically used for intravenous treatment of cutaneous T cell lymphoma. 1.2.2.1.2 SS1P - a recombinant immunotoxin targeting mesothelin The first mesothelin directed agent to enter the clinic was the recombinant immunotoxin (RIT) SS1P. The safety and tolerability of SS1P was determined in two phase I clinical trials in patients. In one trial SS1P was given as a bolus infusion over 30 minutes and the dose-limiting toxicity was pleuritis.[12] In another trial SS1P was given as a continuous infusion for 10 days. Dose-limiting side effects were very similar to the bolus infusion trial.[13] In neither trial were major responses observed, although there was shrinkage of small volume disease in some patients. Phase I studies established a maximum tolerated dose (MTD) of 45 mcg/kg for SS1P given every other day for three total doses per cycle. Dose limiting toxicities (DLTs) were pleuritis, an expected on-target off-tumor toxicity caused by SS1P-induced inflammation of the normal pleura, and secondly, vascular leak syndrome (VLS).  
These early trials demonstrated that the efficacy of SS1P is limited by its immunogenicity. AntiSS1P neutralizing antibodies (Nabs) formed after one cycle in ~90% of patients preventing effective drug exposure in subsequent cycle.[12, 14] Development of human antibody response to immunotoxins in patients with solid tumors has been a significant impediment to their clinical development and previous efforts to limit their immunogenicity by treatment with single agents such as steroids, cyclophosphamide, cyclosporine or rituximab have not been successful.[15-17] However, we have recently shown that when SS1P was administered with a lymphocyte-depleting conditioning regimen of pentostatin and cyclophosphamide, it delayed the development of antiSS1P antibodies in immunocompetent mice.[18]
This strategy of using pentostatin and cyclophosphamide to prevent human antibody response to SS1P was evaluated in a pilot study of heavily pre-treated chemotherapy refractory mesothelioma patients. Eleven patients were enrolled in the study and received pentostatin and cyclophosphamide prior to SS1P administration. This regimen was well tolerated and no patient developed opportunistic infections. As predicted this regimen decreased the development of human antibodies to SS1P; only two of ten patients developed antibodies after cycle 1 of therapy, which was significantly better than in prior clinical trials where about 90% of patients developed antibodies after one cycle of therapy.[19] Remarkably, three of the ten evaluable patients with extensive tumor burden had durable partial response and two of these patients had complete metabolic response by PET scan. All three patients were alive after 27, 25 and 22 months of starting therapy. In addition, two patients who did not initially respond to SS1P had a dramatic anti-tumor response when treated with chemotherapy to which they had not previously responded. SS1P plus pentostatin/cyclophosphamide regimen is currently being evaluated in more patients with mesothelioma. Major tumor shrinkage in the neck and abdomen of a patient with metastatic peritoneal mesothelioma treated with SS1P in combination with pentostatin and cyclophosphamide is shown in Figure 1. These results provide a proof of principle that RITs can have meaningful clinical efficacy in patients with advanced solid tumors. A new generation of immunotoxin molecules with reduced immunogenicity and non-specific toxicity has been developed through protein engineering techniques.  
Figure 1. **Representative response to SS1P in a mesothelioma patient**
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1.2.3 lmb-100 1.2.3.1 rationale for

The clinical use of SS1P, and of immunotoxins in general, has been hampered mainly by their high immunogenicity which limits the number of effective treatment cycles that patients can receive.  
LMB-100 (see Figure 2 for structure) is a next generation PE-fusion protein that has been proteinengineered to maximally reduce its immunogenicity by:
Using a fully humanized Fab fragment derived from the anti-mesothelin antibody SS1 for tumor targeting  
Substituting the bulk of domain II (residues 251–273 and 284–394 of native PE) by an extended furin cleavable linker whose sequence is devoid of any T cell neo-epitopes Deimmunizing domain III of PE, which has the catalytic activity for ADP-ribosylation by introducing 7 point mutations that silence B- and T-cell epitopes
Classical PE-based immunotoxins, such as SS1P, contain a 38 kD fragment of the exotoxin encompassing the so-called translocation domain II and the catalytic domain III. Omission of the domain II from LMB-100 has not only removed a highly immunogenic 14 kD portion of PE that contains the main T-cell epitopes,[20] but has also resulted in reduced incidents of VLS in animal models of VLS.[21] 1.2.3.2 Development of LMB-100: LMB-100 (previously RO6927005 and RG7787) is a next generation anti-mesothelin RIT developed in NCI's Laboratory of Molecular Biology in collaboration with Roche (Figure 2).  
LMB-100 contains a newly engineered PE fragment that has improved activity against most mesothelin-expressing cancer cell lines in vitro, and is also much less toxic than SS1P in preclinical models. This improved therapeutic window allows administration of three to eight times the dose of RIT to mice, rats and monkeys compared to SS1P. The new PE contains modifications specifically designed to reduce immunogenicity of the molecule. This includes deletion of a 14 kD sequence that precedes the catalytic domain and seven point mutations within the catalytic domain itself. These changes ablate the major human B cell epitopes within the molecule and also the most antigenic T cell epitope.[21, 22] The anti-mesothelin targeting region of LMB-100 uses a humanized Fab fragment instead of the smaller dsFv fragment used in SS1P. This increases molecular weight of the RIT above the threshold required to prevent filtration by the kidney and increases half-life.  
  
1.2.3.3 Nonclinical Studies 1.2.3.3.1 Nonclinical Pharmacology In vitro LMB-100 inhibited viability of a variety of mesothelin-positive cancer cell lines at effective concentrations typically around 14 pM (~1 ng/mL). The cytotoxic potency of LMB-100 varied between 0.35 ng/mL in primary mesothelioma cells (RH21) and 15.7 ng/mL in an adenosquamous lung carcinoma cell line (H596). Binding studies showed that while the Fab fragment did not bind to mouse or rat mesothelin, the binding affinities to cynomolgus and human mesothelin were identical. In agreement with this, LMB-100 induced apoptosis in mesothelinpositive primary cynomolgus pericardial cells and significantly impaired viability of HEK293 cells transfected with human mesothelin, but not of rat mesothelin transfected or untransfected HEK293 cells. In addition, control experiments showed that free PE24 was 1001000 fold less potent on mesothelin-positive target cell lines, confirming low cytotoxic potential of PE24 lacking a targeting moiety. LMB-100 showed broad activity against different mesothelin-expressing cancer cell lines and patient derived xenograft models.[23, 24] Animal studies demonstrated that a single cycle of LMB-100 treatment given at an optimal dose of approximately 2 mg/kg, 3  per week, every other day (QOD) achieved tumor regressions in subcutaneous xenografts of adenosquamous lung carcinoma (H596) in severe combined immunodeficient (SCID) beige mice. Three consecutive treatment cycles, given with 1 week breaks in between, led to massive shrinkage of large tumors with an average initial volume of 600 mm3. Tumor regressions in monotherapy were also achieved when treating subcutaneous xenografts of mesothelioma (NCI-H226), gastric (MKN-28), and triple negative breast (HCC70) cancer cell lines in athymic nude mice. Highly synergistic antitumor efficacy was observed in combination therapy with paclitaxel when treating subcutaneous xenografts of the recombinant high mesothelin expressing A431/H9 cell line or the pancreatic cancer cell line KLM1. Synergy was also observed in the HCC70 and MKN-28 cell lines. These results support evidence that LMB100 in monotherapy or in combination with standard chemotherapies may provide clinical benefit to patients with cancer.
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1.2.3.3.2 pharmacokinetics in animals

The pharmacokinetics (PK) of LMB-100 were tested in cynomolgus monkeys following a single IV administration at doses ranging from 0.03 mg/kg to 0.3 mg/kg. Two different enzyme-linked immunosorbent based formats were used for analyzing plasma levels of LMB-100; free and total drug assay (where the total drug assay was the sum of free LMB-100 and LMB-100 complexed with binding molecules). LMB-100 showed a relatively rapid plasma clearance and a volume of distribution at steady-state similar to the plasma volume. Within the dose range tested, non-linear PK was observed for free drug with an extended half-life at higher doses (mean terminal half-life approximately 0.6 hours at 0.3 mg/kg compared to 0.3 hours at 0.03 mg/kg) suggesting saturation of MSLN-mediated clearance pathways. Clearance of total drug was consistently lower than that for free drug implying the presence of soluble binding partners such as soluble mesothelin and ADAs. Induction of anti-drug antibodies (ADA) responses was frequently detectable in all dose groups tested. Overall, given the limited predictive value of immunogenicity reactions in animals to human, a risk for immunogenicity in humans cannot be excluded. Toxico-kinetics after repeated IV dosing in cynomolgus monkeys demonstrated an increase in total exposure in a dose proportional manner between 0.1 mg/kg and 3.0 mg/kg. No accumulation was observed over 5 consecutive days of treatment or over two dosing cycles with 3  per week dosing. Almost all monkeys developed ADAs upon treatment, while induction of high ADA levels impaired the exposure of free drug. In some cases the induction of ADAs may have induced a slight increase in exposure The relationship between systemic drug exposure and anti-tumor activity of LMB-100 was investigated on human lung cancer NCI-H596 xenograft growth in female SCID beige mice. Free and total drug profiles were similar in mice. Modeling estimated a plasma concentration of 6800 ng/mL ( 36%) to trigger a half maximal rate of tumor regression. Concentrations of LMB-100 above this level resulted in potent tumor regression after dosing. Normalized for exposure, SS1P was found to be ~3-fold more potent than LMB-100 in terms of tumor growth inhibition.
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1.2.3.3.3 toxicology and safety pharmacology

The toxicological profile of LMB-100 was assessed after repeated intravenous administration to cynomolgus monkeys, the only relevant species, for a maximum of 5 daily doses for one week or 2 cycles with QOD  3 dosing, separated by a 9 day dosing free period. Four daily doses of 3 mg/kg exceeded the maximum tolerated dose with animals being found in moribund condition, indicated by clinical signs of hypoactivity, hunched posture, ataxia, and tremors. There were no histopathological changes to account specifically for the deteriorating physical condition of these animals. Histopathological findings such as kidney tubular degeneration/regeneration and changes at serosal-lining tissues were observed at lower doses as well. In general, administration of LMB100 resulted in both on- and off-target toxicities.  
On-target effects were observed on serosal-lining tissues, consistent with high expression of mesothelin. Mesothelium hypertrophy accompanied by subpleural cellular hypertrophy and serosal fibrin exudate was observed in the lung at doses  1 mg/kg. Mesothelium hypertrophy also occurred in heart (epicardium), spleen, and stomach. Off-target or non-specific toxicity included degeneration/regeneration of kidney tubular epithelium after repeated doses of  0.3 mg/kg. Local inflammatory findings at the injection sites were observed after administration of LMB-100 in several studies. Clinically, reddening of the skin, swelling, and skin being warm to touch or flaky injection sites were reported. In the 2-cycle GLP study (3 intermittent doses over a 5-day period, 9-days apart), impaired movement of animals from all dose groups was likely related to injection site findings and an overall inflammatory profile. One female at 1 mg/kg was sacrificed early on Day 4 after 2 doses due to severe clinical signs most likely attributed to inflammatory changes at injection sites resulting in moribundity of the animal. Clinically observed inflammatory changes correlated with histopathological changes such as hemorrhages and/or acute inflammation at the injection sites and clinical pathology changes consistent with an overall inflammatory profile (increases in monocytes, neutrophils, CRP, and haptoglobin). Microscopic changes reversed completely after the 4-week recovery period in the 2-cycle GLP study. The Highest Non Severely Toxic Dose in this study was 0.3 mg/kg, which resulted in a mean AUC for total drug of 16.0 µg·h/mL (study day 1, preliminary data). In a subsequent 1 cycle GLP study (QOD  3 dosing),
markedly reduced Injection site findings were observed after administration of a batch with reduced levels of product related modifications of LMB-100. In this study, the HNSTD was 1 mg/kg, resulting in an AUC for total drug of 27.4 and 23.6 µg·h/mL after the first and third dose (preliminary data). The potential of LMB-100 to induce off-target vascular leak in lungs was assessed in female Wistar rats. Mild perivascular edema was reported microscopically, but did not correlate with macroscopic or serum chemistry findings consistent with VLS. Ultrasound evaluation in the NHP GLP study revealed minimal accumulation of pericardial fluid with limited biological significance at the highest dose of 1 mg/kg. No appreciable accumulation of pleural fluid was observed at necropsy.  
In vitro evaluation of LMB-100 in human whole blood assay indicated a low risk for cytokinemediated infusion related reaction (IRR)/cytokine release syndrome (CRS) upon first administrations. LMB-100 caused no hemolysis when added to human peripheral blood up to the highest concentrations of 0.5 mg/L. 1.2.3.3.4 LMB-100 (formerly RG7787) plus *nab-*paclitaxel (Abraxane) against mesothelioma patient derived xenografts (PDX)
Since our prior studies have shown remarkable synergy between immunotoxins and taxanes we evaluated the efficacy of nab-paclitaxel with LMB-100. We chose to use nab-paclitaxel instead of paclitaxel since clinical trials of paclitaxel have shown no activity in patients with mesothelioma.[25, 26] Nab-paclitaxel is an albumin bound paclitaxel that has distinct pharmacologic properties compared to paclitaxel including greater uptake by and retention within tumor, which could make it efficacious to treat solid tumors.[27] Potential toxicities of nabpaclitaxel are listed in section 14.2.2 and product package insert.  
Primary mesothelioma cells are much more sensitive to LMB-100 than SS1P: Primary mesothelioma cell lines NCI-Meso16, NCI-Meso19, NCI-Meso21 and NCI-Meso29 established from patients were evaluated for sensitivity to the anti-mesothelin immunotoxins.[28]
All four of these cell lines are sensitive to killing by LMB-100 with IC50 of 0.3 to 10 ng/ml with NCI-Meso21 being most sensitive with an IC50 of 0.3 ng/mL (Figure 3). Surprisingly, most of these primary mesothelioma cells except NCI-Meso16 were 2 to 9-fold more sensitive to LMB100 than SS1P. In case of nab-paclitaxel, NCI-Meso16, NCI-Meso21 and NCI-Meso29 were sensitive to nab-paclitaxel with IC50 of 10, 20 and 25 ng/ml respectively, while as NCI-Meso19 was resistant with IC50 >100ng/mL (Figure 3).  
The *in-vitro* activity of LMB-100 in combination with *nab*-paclitaxel was evaluated using NCIMeso16, NCI-Meso21 and NCI-Meso29 cell lines since they are sensitive to both LMB-100 and nab-paclitaxel. The combination of LMB-100 and nab-paclitaxel resulted in decreased cell viability compared to untreated cells, LMB-100 or *nab*-paclitaxel alone treated cells for all three cell lines. This was especially pronounced for NCI-Meso16 cells that had an IC50 of 0.01 for LMB100 plus nab-paclitaxel compared to their single agent IC50 of 10 ng/ml (Figure 4A). There was also a sharp increase in the % of apoptotic cells when nab-paclitaxel and LMB-100 were used in combination vs. nab-paclitaxel or LMB-100 alone (Figure 4B). Figure 4: **In-vitro synergy between LMB-100 and nab-paclitaxel (A) as well as induction of**  
 apoptosis (B) against NCI-Meso16 cells. LMB-100 plus nab-paclitaxel have marked anti-tumor efficacy against mesothelioma PDX models: Figure 5 describes the anti-tumor activity of LMB-100, nab-paclitaxel or combination of the two against NCI-Meso16 tumor xenografts. As shown, treatment with LMB-100 or nab-paclitaxel slowed tumor growth but after treatment was stopped the tumors rapidly increased in size. However, in mice that were treated with LMB-100 plus nab-paclitaxel there was complete tumor regression in all mice with 5 /7 mice being tumor free when the experiment was terminated on day 142. Evaluation of tumors in mice treated with LMB-100 plus nab-paclitaxel showed increased apoptosis, decreased Ki-67 staining and decreased mesothelin positive cells compared to LMB100 or nab-paclitaxel alone treated mice. Similar anti-tumor efficacy of LMB-100 plus nabpaclitaxel was also seen against NCI-Meso21 and NCI-Meso29 PDX models. These pre-clinical studies provide strong rationale for clinical trial of LMB-100 alone or with nab-paclitaxel to treat patients with mesothelioma. Figure 5: Anti-tumor efficacy of LMB-100 plus nab**-paclitaxel against**  
  
mesothelioma PDX NCI Meso-16.  
Serum mesothelin is a biomarker of tumor response in mesothelioma PDX models: Since the NCI-Meso16, NCI-Meso21 and NCI-Meso29 tumors highly express mesothelin we hypothesized that these tumors could release human mesothelin in mouse blood and therefore measuring the levels could be used to assess response to therapy. As shown in Figure 6 in mice with NCI-Meso16 tumors, the serum mesothelin levels increased in the control group and decreased in the LMB-100 and nab-paclitaxel group and became undetectable in the combination group. There was excellent correlation between tumor size and serum mesothelin levels in these mice (r=0.89; p<0.0001). Similarly, in the NCI-Meso21 and NCI-Meso29 tumor model there was very good correlation between tumor size and serum mesothelin in the different treatment groups. These results provide pre-clinical evidence that serum mesothelin is a robust marker of tumor response in mesothelioma and supports earlier clinical observations by us and others that serum mesothelin levels correlate with radiologic tumor response in patients.[29-31]
Figure 6: Correlation between tumor size, tumor response and serum mesothelin for NCIMeso16 tumors.
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1.2.3.3.4.1 nab-paclitaxel dose justification

The standard dose for nab-paclitaxel in non small cell lung cancer is 100 mg/m2 on days 1, 8 and 15 of each 21 day cycle. The same dose will be used on this study; however, administration will be less frequent (days 1 and 8 of each 21-day cycle) in order to decrease possible toxicity due to the combination. Given the expected side effects of nab-paclitaxel including peripheral sensory neuropathy, fatigue and grade 3/4 neutropenia we have reduced the dose frequency of nabpaclitaxel by 33% to 100 mg/m2 given on days 1 and 8 of each 21-day cycle.  
1.2.3.4 Clinical testing of single agent LMB-100 (Roche Study) Initial clinical testing of LMB-100 was performed by Roche in a multi-center international first in human trial (NCT02317419). The primary objective of the Phase I study was to define the safety and tolerability (including the MTD) and pharmacokinetics of the drug in participants with MSLNexpressing metastatic or locally advanced solid tumors for whom no standard therapy was available. Secondary objectives included determination of the RP2D and schedule, exploration of preliminary anti-tumor activity by assessing objective response rate (ORR) and disease control rate (DCR), and assessment of pharmacodynamic effects. A total of 15 participants were enrolled onto the study before termination. Median age of participants was 60.8 yrs and 53.3% were female. All participants had received prior anti-cancer therapy for their tumors. Enrolled participants had advanced mesothelioma (7), ovarian cancer (3), pancreatic cancer (3), and gastroesophageal cancer (2). Tumors from 13 of the 15 participants treated had moderately to strongly positive MSLN expression as measured by central IHC analysis.  
LMB-100 was administered intravenously on Days 1, 3 and 5 of a 21 day treatment cycle. No premedications were given. Treatment was initiated at the MTD of SS1P, 45 mcg/ kg. Five different dose levels were tested (see Table 1). Dose limiting toxicity (DLT) was reached at 250 mcg/kg, with 2 of 4 participants treated at this dose level experiencing vascular leak syndrome (grade 2 and grade 4). Additional toxicities were associated with this dose level. At this point, a sixth cohort receiving 200 mcg/kg of study drug was enrolled, however, the study was terminated by the company before the two accrued participants completed cycle 1 of therapy. Therefore the single agent MTD was not determined.  
| Dose (mcg/kg)                                    | No. of patients   | Pts with DLT   |
|--------------------------------------------------|-------------------|----------------|
| 45                                               | 1                 | 0              |
| 65                                               | 1                 | 0              |
| 100                                              | 3                 | 0              |
| 170                                              | 4                 | 0              |
| 200                                              | 2                 | NE             |
| 250                                              | 4                 | 2              |
| DLTs were vascular leak syndrome and proteinuria |                   |                |  
NE, Study terminated before DLT assessment period was complete and patients only received single dose of LMB-100
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1.2.3.4.1 lmb-100 adverse events

Overall, 14 participants (93.3%) experienced at least one AE. The most common AEs were hypoalbuminemia (60.0%), fatigue (53.3%), peripheral edema (53.3%), nausea (46.7%), pyrexia (40.0%), decreased appetite (33.3%), dyspnea (33.3%), and myalgia (33.3%). SAEs included vascular leak syndrome, pyrexia, atrial flutter, infusion related reaction, arthritis, glomerulonephritis minimal lesion and dyspnea. No participants experienced an AE that led to withdrawal of study treatment. Four participants experienced a total of 8 infusion-related reactions that were independent of drug dose level. All of these AEs were non-serious and resolved within approximately 1 hour of onset. Pre-medication for infusion reaction was administered to these participants prior to subsequent doses of LMB-100. Two suspected Type III hypersensitivity reactions were observed. These consisted of arthritis (1 patient) and rash with fever (1 patient), both of which were fully reversible. When other AEs attributed to the study drug are presented by dose level of drug, it becomes clear that toxicity was strongly associated with the 250 mcg/kg dose level at which DLT was reached. Two of four patients treated at 250 mcg/kg experienced serious
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1.2.3.4.1 lmb-100 adverse events

VLS which manifested with hypotension, respiratory compromise, serosal membrane reaction and hyponatremia as well as the hypoalbuminemia and edema that can be seen with mild VLS. Other symptoms associated with the DLT dose were fatigue, nausea, vomiting, decreased appetite and mild elevation of transaminases. Table 2 summarizes any adverse event related to LMB-100 while as Table 3 summarizes grade 3 or 4 toxicity related to LMB-100. As shown in Table 3 most grade 3 or 4 toxicity were seen in patients treated at 250 mcg/kg dose level. However, patients receiving LMB-100 doses less than 250 mcg/kg had mainly grade 1-2 adverse events except 1 patient having grade 3 arthritis and 1 patient with grade 3 anemia.  
| Table 2: Adverse Events attributed to LMB-1001 Dose (mcg/kg) 45 65   | 100   | 170   | 250   | ALL   |    |    |
|----------------------------------------------------------------------|-------|-------|-------|-------|----|----|
| # of patients treated                                                | 1     | 1     | 3     | 4     | 4  | 13 |
| Vascular Leak                                                        | -     | -     | 1     | 1     | 2  | 4  |
| grade 3 or 4 vascular leak                                           | -     | -     | -     | -     | 1  | 1  |
| hypotension                                                          | -     | -     | -     | -     | 2  | 2  |
| hypoalbuminemia                                                      | -     | 1     | 3     | 2     | 3  | 9  |
| peripheral edema                                                     | 1     | 1     | 3     | 2     | 4  | 11 |
| facial edema                                                         | -     | 1     | 2     | -     | 1  | 4  |
| weight gain                                                          | -     | -     | 1     | -     | 1  | 2  |
| hyponatremia                                                         | -     | -     | -     | -     | 3  | 3  |
| hypophosphatemia                                                     | -     | -     | -     | -     | 1  | 1  |
| dyspnea                                                              | -     | 1     | -     | -     | 2  | 3  |
| Infusion related reaction                                            | -     | -     | 2     | -     | 2  | 4  |
| Constitutional  fatigue                                              | -     | -     | -     | 1     | 4  | 5  |
| asthenia                                                             | -     | -     | 3     | -     | -  | 3  |
| fever                                                                | 1     | -     | 2     | 1     | 2  | 6  |
| Musculoskeletal myalgia                                              | -     | -     | 1     | 1     | 2  | 4  |
| arthralgia                                                           | 1     | -     | -     | -     | 1  | 2  |
| arthritis                                                            | -     | -     | 1     | -     | -  | 1  |
| muscle spasm                                                         | -     | -     | -     | 1     | -  | 1  |
| Cardiac pericardial effusion                                         | -     | -     | -     | -     | 1  | 1  |
| Atrial flutter                                                       | -     | -     | -     | 1     | -  | 1  |
| Renal Disorders glomerulonephritis minimal                           | -     | -     | -     | 1     | -  | 1  |
| proteinuria                                                          | -     | -     | -     | 1     | 1  | 2  |
| Creatinine increase                                                  | -     | -     | -     | -     | 2  | 2  |
| Gastrointestinal decreased appetite                                  | -     | 1     | -     | 1     | 2  | 4  |
| nausea                                                               | 1     | -     | 1     | 1     | 4  | 7  |
| abdominal pain                                                       | -     | 1     | -     | 1     | 1  | 3  |  
| Dose (mcg/kg)                                                                                                       | 45   | 65   | 100   | 170   | 250   | ALL   |
|---------------------------------------------------------------------------------------------------------------------|------|------|-------|-------|-------|-------|
| # of patients treated                                                                                               | 1    | 1    | 3     | 4     | 4     | 13    |
| diarrhea                                                                                                            | 1    | -    | 1     | -     | -     | 2     |
| vomiting                                                                                                            | -    | -    | -     | -     | 2     | 2     |
| abdominal distension                                                                                                | -    | 1    | -     | -     | -     | 1     |
| constipation                                                                                                        | -    | -    | -     | 1     | -     | 1     |
| dyspepsia                                                                                                           | -    | -    | -     | 1     | -     | 1     |
| AST increase                                                                                                        | -    | -    | -     | -     | 2     | 2     |
| Hematologic anemia                                                                                                  | -    | -    | -     | 1     | -     | 1     |
| decreased lymphocytes                                                                                               | -    | -    | -     | -     | 1     | 1     |
| Total Grade 3 or greater                                                                                            | -    | -    | 2     | 1     | 2     | 5     |
| Orange highlighting indicates that one of the patients experienced a grade 3 or 4 toxicity in this category. Please |      |      |       |       |       |       |  
Orange highlighting indicates that one of the patients experienced a grade 3 or 4 toxicity in this category. Please note that there was only 1 patient who experienced a high grade toxicity of each type.  
1 adapted from Clinical Study Report No 1066017 from Roche dated December 2015, "Summary of Adverse Events Related to Study Medication, Safety-Evaluable Patients Protocol: BP29387" See pages 175-193 of the report."
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table 3 grade 3 or

| Dose (mcg/kg)                                                                                              | 45   | 65   | 100   | 170   | 250   | ALL   |
|------------------------------------------------------------------------------------------------------------|------|------|-------|-------|-------|-------|
| # of patients treated                                                                                      | 1    | 1    | 3     | 4     | 4     | 13    |
| Vascular leak (gr 4)                                                                                       | -    | -    | -     | -     | 1     | 1     |
| Hyponatremia (gr 3)                                                                                        | -    | -    | -     | -     | 1     | 1     |
| Anemia (gr 3)                                                                                              | -    | -    | -     | 1     | -     | 1     |
| Decreased lymphocytes (gr 3)                                                                               | -    | -    | -     | -     | 1     | 1     |
| Dyspnea (gr 3)                                                                                             | -    | -    | -     | -     | 1     | 1     |
| Infusion-related reaction (gr 3)                                                                           | -    | -    | 1     | -     | -     | 1     |
| Arthritis (gr 3)                                                                                           | -    | -    | 1     | -     | -     | 1     |
| 1 adapted from Clinical Study Report No 1066017 from Roche dated December 2015, "Summary of Adverse Events |      |      |       |       |       |       |
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1.2.3.4.2 anti-drug antibodies (adas) and

Twelve participants were evaluable for efficacy. The best confirmed overall response was stable disease in 3 participants. A Roche-developed ELISA test was used to retrospectively assess antidrug antibody (ADA) titers. Four of 13 patients had detectable ADAs at study enrollment while the remaining participants did not; however, the remaining 10 participants developed detectable ADAs by the end of Cycle 2. (Figure 7, Table 4 and Table 5). All evaluable participants achieved expected serum drug levels during the first cycle of treatment. Six of 7 participants without preexisting ADAs achieved effective drug levels during the second cycle, while 0 of 2 participants with pre-existing ADAs did. One of 3 participants that received a third cycle of treatment also achieved effective drug levels during this cycle. A positive test for ADAs did not definitively predict poor blood levels in the subsequent cycle (see patient 1101 in Table 5, who had very good LMB-100 blood levels despite the presence of ADA). In summary the data regarding LMB-100 ADA and LMB-100 blood levels show:
Presence of ADA is not predictive of ability to achieve measurable LMB-100 concentration in the serum, which is the most important parameter for drug efficacy.  
Patients who have presence of pre-existing ADA are able to achieve good LMB-100 blood levels during cycle 1 (4/4 patients), but may not do so during cycle 2 (2/2 patients with undetectable blood levels) although the numbers are small.  
Patients who do not have presence of pre-existing ADA are able to achieve good LMB100 blood levels during cycle 1 (7/7 patients) as well as cycle 2 (6/7 patients).  
Very few patients have been to determine LMB-100 blood levels during cycle 3 and beyond. However, 1/3 patients had good LMB-100 blood levels that were in fact higher than those achieved during cycle 1 and 2 despite the presence of ADA. In addition, another patient had low but detectable blood levels during cycle 3. More patients need to be treated to determine what percent of patients will achieve good blood levels during cycle 3 and beyond.
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table 4 effect of ada

The table shows patients treated at different dose levels of LMB-100; ADA prior to start of each cycle and LMB-100 blood levels as Cmax (ng/ml). In addition, the change in LMB-100 Cmax concentration (as percent increase or percent decrease) during cycle 2 and 3 is shown as dC1/dC2 and dC1/dC3 respectively. Please note that - means the PK assay was not performed.  
| Cycle 1                                            | Cycle 2   | Cycle 3   |         |        |         |        |        |         |        |
|----------------------------------------------------|-----------|-----------|---------|--------|---------|--------|--------|---------|--------|
| Patien                                             | Dose      | ADA       | Cmax    | ADA    | Cmax    | dC1/dC | ADA    | Cmax    | dC1/dC |
| t                                                  | (mcg/kg)  | (Day1)    | (ng/ml) | (Day1) | (ng/ml) | (%)    | (Day1) | (ng/ml) | (%)    |
| 1002                                               | 65        | 0         | 1150    | 8100   | 711     | -38    | -      | -       | -      |
| 1401*                                              | 100       | 0         | 1790    | 0      | 1610    | -10    | 24300  | 267     | -85    |
| 1402                                               | 100       | 0         | 1650    | 0      | 1360    | -18    | 8100   | 0       | -100   |
| 1101*                                              | 170       | 0         | 2760    | 300    | 3950    | 43     | 900    | 3490    | 26     |
| 1202                                               | 170       | 0         | 3040    | 0      | 1940    | -36    | -      | -       | -      |
| 1301                                               | 170       | 0         | 3430    | 72900  | 527     | -85    | -      | -       | -      |
| 1403                                               | 170       | 0         | 1930    | 900    | 1550    | -20    | -      | -       | -      |
| 1102*                                              | 250       | 0         | 5480    | 0      | 4770    | -13    | -      | -       | -      |
| 1302                                               | 250       | 0         | 4340    | -      | -       | -      | -      | -       | -      |
| * Patient's treatment stopped due to study closure |           |           |         |        |         |        |        |         |        |
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table 5 effect of ada

The table shows patients treated at different dose levels of LMB-100; ADA prior to start of each cycle and LMB-100 blood levels as Cmax (ng/ml). In addition, the change in LMB-100 Cmax concentration (as percent increase or percent decrease) during cycle 2 and 3 is shown as dC1/dC2 and dC1/dC3 respectively. Please note that - means the PK assay was not performed.  
| Cycle 1   | Cycle 2   |         |        |         |      |         |
|-----------|-----------|---------|--------|---------|------|---------|
| Patient   | Dose      | ADA     | Cmax   | ADA     | Cmax | dC1/dC2 |
| (mcg/kg)  | (Day1)    | (ng/ml) | (Day1) | (ng/ml) | (%)  |         |
| 1001      | 45        | 2700    | 620    | 196830  | 0    | -100    |
| 1201      | 100       | 8100    | 495    | 196830  | 0    | -100    |
| 1501*     | 250       | 2700    | 3730   | 196830  | -    | -       |
| 1003      | 250       | 900     | 2960   | 196830  | -    | -       |  
* Patient's treatment stopped due to study closure  
1.2.3.5 Clinical testing of single agent LMB-100 (Current Study - 16C0127 as of 8/15/17)
1.2.3.5.1 Arms A1 and A2  
  
Figure 8: Serum Creatinine for Patients 1, 2 and 3 treated at 170 µg/kg As of December 20, 2016 when Arms A1 and A2 (single agent LMB-100) were closed and Arms B1 and B2 (LMB-100 + nab-paclitaxel) were established ten patients were enrolled on the study. Out of these 10 patients 7 had peritoneal mesothelioma and 3 pleural mesothelioma; 6 female and 4 male and median age was 62 years old. The first 3 patients were treated at dose level 1 i.e. 170 mcg/kg with patient \#1 initiating treatment on July 28, 2016. All three patients at this dose level during cycle 1 had grade 1 or 2 increase in serum creatinine (Figure 8). Since increase in serum creatinine was a common toxicity pattern at this dose level it was defined as DLT as per protocol established criteria (per bullet one under Other Toxicities in section 3.1.1). The protocol was subsequently amended (Amendment B dated 08/23/16) to allow the treating these three patients at dose level -1 (140 mcg/kg) during cycle 2-4 though the protocol prior to the amendment would have allowed retreatment at 170 mcg/kg as long as they met inclusion criteria for the study especially adequate renal function, defined in the protocol as creatinine clearance (by Cockcroft Gault formula) >50 mL/min. However, we felt it would be safer to re-treat these patients at dose level-1 instead of dose level 1 and adjusted the dose modification section (3.3.1) of the protocol accordingly as part of Amendment B. All subsequent patients were treated at 140 mcg/kg. Patients 001-003 have completed 4 cycles of LMB-100 treatment (LMB-100 170 mcg/kg during cycle 1 and LMB-100 140 mcg/kg during cycle 2-4) and seven additional patients have been enrolled at dose level -1 (140 mcg/kg). No DLT's have been observed at the LMB-100 dose level of 140 mcg/kg. Therefore, the recommended phase of single agent LMB-100 is 140 mcg/kg given every 3 weeks.  
Grade 2 infusion reactions were seen in 5 patients at some point during the treatment with LMB100. In 2 patients it was seen during cycle 2 and 3 patients during cycle 3 and cycle 4 of treatment.  
In most case the infusion reaction was managed by administration of dexamethasone and increasing infusion duration as per protocol. However, in two patients who had infusion reaction during cycle 4 of LMB-100 the treatment was discontinued. No serious adverse events were observed. The most frequently occurring events were hypoalbuminemia (11 events), anemia (6), increased creatinine (4), decreased lymphocytes (4), hyperglycemia (3), dyspepsia (2) and fever (2). There were single occurrences of limb edema, decreased platelets, weight gain and weight loss. Most of the events were grade 2 with the exception of one grade 3 decreased lymphocytes and three grade 3 hyperglycemia. Out of 10 patients treated with LMB-100 alone who have been evaluated for tumor response after cycle 2, 9 had stable disease and 1 had progressive disease.
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1.2.3.5.2 arm b1

As of August 15, 2017, 6 patients have been treated at Arm B1's dose level 1 (i.e. LMB-100: 100 mcg/kg on days 1, 3 and 5 plus nab-paclitaxel: 100 mg/m2 on days 1 and 8 of a 21-day cycle. Of these 6 patients, 1 patient was not evaluable for toxicity since he did not receive day 8 nabpaclitaxel during cycle 1. Of the 5 patients who were evaluable for toxicity we had one DLT (grade 4 myositis/CPK elevation).
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1.2.3.5.3 pharmacokinetics of lmb-100

Free LMB-100 plasma concentrations were measured with a validated ELISA with a lower limit of quantification of 2.1 ng/mL. Doses ranged from 140 mcg/kg - 170 mcg/kg. Samples for pharmacokinetic (PK) analysis were obtained from patients at pre-dose, end of infusion (EOI; 30min post start), and 1 hour, 2 hours, 3 hours, 4 hours and 6 hours post EOI. Concentration data for each dose was plotted over time to assess the impact of increasing anti-drug antibodies (ADAs) that are generated in response to LMB-100 exposure. PK analysis was done for the first five patients treated on the study and is shown in Table 6 below. Measured LMB-100 plasma concentrations were consistent during the first week of treatment, with a near dose-proportional increase in CMAX from 140 mcg/kg to 170 mcg/kg. However, the suspected generation of ADAs greatly reduced LMB-100 exposure by cycle 2. As shown in Table 6, all 10 patients had good LMB-100 blood levels during cycle 1. However only 5 of 10 patients had detectable blood levels during cycle 2. None of the 9 patients who got cycle 3 of LMB-100 had good LMB-100 blood levels during cycle 3; and none of 6 patients had good LMB-100 blood levels during cycle 4.  
| C1D1    | C2D1    | C3D1    | C4D1    |         |         |         |         |      |
|---------|---------|---------|---------|---------|---------|---------|---------|------|
| Patient | Dose    | Cmax    | Dose    | Cmax    | Dose    | Cmax    | Dose    | Cmax |
| (µg/kg) | (ng/mL) | (µg/kg) | (ng/mL) | (µg/kg) | (ng/mL) | (µg/kg) | (ng/mL) |      |
| 1       | 170     | 1991    | 140     | 1091    | 140     | 21      | 140     | 21   |  
| C1D1    | C2D1    | C3D1    | C4D1    |         |         |         |         |      |
|---------|---------|---------|---------|---------|---------|---------|---------|------|
| Patient | Dose    | Cmax    | Dose    | Cmax    | Dose    | Cmax    | Dose    | Cmax |
| (µg/kg) | (ng/mL) | (µg/kg) | (ng/mL) | (µg/kg) | (ng/mL) | (µg/kg) | (ng/mL) |      |
| 2       | 170     | 2286    | 140     | 297     | 140     | 70      | 140     | 21   |
| 3       | 170     | 2760    | 140     | 4.4     | 140     | BQL     | 140     | BQL  |
| 4       | 140     | 1054    | 140     | 681     | 140     | BQL     | N/A     | N/A  |
| 5       | 140     | 1124    | 140     | BQL     | N/A     | N/A     | N/A     | N/A  |
| 6       | 140     | 2584    | 140     | 1099    | 140     | BQL     | 140     | BQL  |
| 7       | 140     | 3118    | 140     | 2450    | 140     | BQL     | 140     | BQL  |
| 8       | 140     | 1721    | 140     | BQL     | 140     | BQL     | 140     | BQL  |
| 9       | 140     | 2584    | 140     | 1099    | 140     | BQL     | 140     | BQL  |
| 10      | 140     | 3118    | 140     | 2450    | 140     | BQL     | 140     | BQL  |  
These results are in agreement with the Roche phase I clinical trial. It is also clear that administration of LMB-100 beyond cycle 2 is unlikely to result in meaningful clinical benefit since there are no detectable blood levels during cycle 3 and 4. Based on the result of the data from the first 5 patients, as of Amendment C (dated 03/09/17) LMB-100 will only be administered during cycles 1 and 2 in patients enrolled subsequent this amendment (Arms B1 and B2). Previously enrolled subjects will continue to receive 4 cycles of LMB-100 as PK data are not yet complete.
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1.2.4 comparison of toxicity and

LMB-100 was safely administered to four patients at 170 mcg/kg without DLT. This is nearly four times the maximum tolerated dose (MTD) of SS1P. Most patients without pre-existing ADAs were able to achieve effective drug levels of LMB-100 for 2 cycles, unlike what has been seen previously with SS1P (see Figure 2 and Figure 9). These data demonstrate that LMB-100 is both less toxic and less immunogenic than SS1P. Figure 9: The ability to achieve good drug levels during cycle 2 is shown for patients who were treated on a phase I trial of single agent SS1P[12] and those treated with LMB-100. Only 9% of patients treated with SS1P had good blood levels during cycle 2 versus 86% for  
 patients treated with LMB-100 (p>0.001 by two tailed Fisher's exact test).
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1.2.5 rationale for the study

There is clearly an unmet need to identify improved therapies for patients with malignant mesothelioma. LMB-100 is a reduced immunogenicity anti-mesothelin RIT developed in NCI's Laboratory of Molecular Biology in collaboration with Roche. Mesothelin is expressed in over 95% of epithelioid subtype of malignant mesothelioma. Limited dose escalation completed to date indicates safety and provides preliminary data on immunogenicity. Identification of the recommended phase 2 dose (RP2D), now established at 140 mcg/kg and preliminary assessment of its monotherapy efficacy will inform future studies of LMB-100 in combination with other agents.  
As of Amendment C, we also began to evaluate the efficacy and tolerability of 2 cycles of LMB100 at the established RP2D in combination with nab-paclitaxel (Arm B) in order to explore the synergy between the agents as addressed in section 1.2.3.3.4. Initially, we planned to study 2 escalating dose levels of LMB-100, 100 mcg/kg (dose level 1) and 140 mcg/kg (dose level 2) with a stable dose of nab-paclitaxel (100 mg/m2). However, as of Amendment F, it was determined that that the highest dose level tested would be dose level 1 based on data from a companion protocol (16C0128) in which LMB-100+nab-paclitaxel is used the treatment of patients with pancreatic cancer. In this study, 2 of 6 subjects treated with an LMB-100 dose of 100 mcg/kg and a nabpaclitaxel dose 125 mg/ m2 experienced DLT. Therefore, we have decided not to evaluate dose level 2. Dose de-escalation may occur if more than 1 DLT occurs at the initial dose level,
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2 eligibility assessment and enrollment

2.1.1 Inclusion Criteria (All Arms) 2.1.1.1 Histologically confirmed epithelial or biphasic mesothelioma not amenable to potentially curative surgical resection. However, patients with biphasic tumors that have a ≥ 50%
sarcomatoid component will be excluded. The diagnosis will be confirmed by the Laboratory of Pathology, CCR, NCI.  
2.1.1.2 Archival sample or fresh biopsy or tumor effusion must be available for confirmation of diagnosis.  
2.1.1.3 Patients must have measurable disease per RECIST 1.1. as defined per . (See Section 6.3).  
2.1.1.4 Patients must have had at least one prior chemotherapy regimen that includes pemetrexed and cisplatin or carboplatin. There is no limit to the number of prior chemotherapy regimens received.  
2.1.1.5 The last dose of previous therapy must have occurred at least 3 weeks prior to the start of study therapy. Palliative radiotherapy is allowed up to 2 weeks before the first LMB-100 infusion.  
2.1.1.6 Patients for whom no standard curable therapy exists 2.1.1.7 Age ≥ 18 years. Because no dosing or adverse event data are currently available on the use of LMB-100 alone or in combination with nab-paclitaxel in patients <18 years of age, children are excluded from this study 2.1.1.8 All acute toxic effects of any prior radiotherapy, chemotherapy, or surgical procedure must have resolved to Grade  1, except alopecia (any grade) and < Grade 2 peripheral neuropathy.  
2.1.1.9 ECOG performance status (PS) 0 or1 (Appendix A)
2.1.1.10 Adequate hematological function: neutrophil count of  1.5  109 cells/L, platelet count of  100,000/L (transfusion independent, defined as not receiving platelet transfusions within 7 days prior to laboratory sample), hemoglobin  9 g/dL
2.1.1.11 Adequate Liver function: AST and ALT < 2.5 X upper limit of normal, alkaline phosphatase < 2.5 X upper limit of normal, unless bone metastasis is present (<5 X upper limit of normal) in the absence of liver metastasis.  
2.1.1.12 Bilirubin  1.5 mg/dL (excluding Gilbert's Syndrome, see below).  
2.1.1.13 Patients with Gilbert's syndrome will be eligible for the study. The diagnosis of Gilbert's syndrome is suspected in people who have persistent, slightly elevated levels of unconjugated bilirubin without any other apparent cause. A diagnosis of Gilbert's syndrome will be based on the exclusion of other diseases based on the following criteria:
Unconjugated hyperbilirubinemia noted on several occasions No evidence of hemolysis (normal hemoglobin, reticulocyte count, and LDH)
Normal liver function tests Absence of other diseases associated with unconjugated hyperbilirubinemia 2.1.1.14 Adequate renal function: creatinine < 1.5 mg/dL OR creatinine clearance (by Cockcroft Gault formula Appendix B)  50 mL/min.  
2.1.1.15 Must have serum albumin > 2.5 mg/dL without intravenous supplementation 2.1.1.16 Must have left ventricular ejection fraction > 50% 2.1.1.17 Must have an ambulatory oxygen saturation of > 90% on room air 2.1.1.18 The effects of LMB-100 alone or in combination with nab-paclitaxel on the developing human fetus are unknown. For this reason:
2.1.1.18.1 Women of child-bearing potential (defined as a sexually mature woman who (1) has not undergone hysterectomy [the surgical removal of the uterus] or bilateral oophorectomy
[the surgical removal of both ovaries] or (2) has not been naturally postmenopausal for at least 24 consecutive months [i.e., has had menses at any time during the preceding 24 consecutive months]) must:
Either commit to true abstinence* from heterosexual contact (which must be reviewed on a monthly basis), or agree to use, and be able to comply with, effective contraception without interruption, 28 days prior to starting study therapy (including dose interruptions), while on study medication and for 3 months after the last dose of study therapy; and Have a negative serum pregnancy test (β -hCG) result at screening and agree to ongoing pregnancy testing during the course of the study, and after the end of study therapy. This applies even if the subject practices true abstinence* from heterosexual contact.  
2.1.1.18.2 Men must agree to practice true abstinence* or agree to use a condom during sexual contact with a pregnant female or a female of childbearing potential while participating in the study, during dose interruptions and for 6 months following discontinuation of study therapy, even if he has undergone a successful vasectomy.  
2.1.1.19 Ability of subject to understand and the willingness to sign a written informed consent document.  
* True abstinence is acceptable when this is in line with the preferred and usual lifestyle of the subject. [Periodic abstinence (e.g., calendar, ovulation, symptothermal, post-ovulation methods) and withdrawal are not acceptable methods of contraception].  
2.1.2 Exclusion Criteria (All Arms) 2.1.2.1 Known or clinically suspected CNS primary tumors or metastases including leptomeningeal metastases. History or clinical evidence of CNS metastases unless they have been previously treated, are asymptomatic, and have had no requirement for steroids or enzyme-inducing anticonvulsants in the last 14 days.  
2.1.2.2 Evidence of significant, uncontrolled concomitant diseases which could affect compliance with the protocol or interpretation of results, including significant pulmonary disease other than primary cancer, uncontrolled diabetes mellitus, and/or significant cardiovascular disease (such as New York Heart Association Class III or IV cardiac disease, myocardial infarction within the last 6 months, unstable arrhythmias, unstable angina, or clinically significant pericardial effusion)
2.1.2.3 Active or uncontrolled infections. 2.1.2.4 HIV or active HBV or HCV infection. HIV positive patients will be excluded due to a theoretical concern that the degree of immune suppression associated with the treatment may result in progression of HIV infection.  
2.1.2.5 Patients with prior pneumonectomy 2.1.2.6 Any other diseases, metabolic dysfunction, physical examination finding, or clinical laboratory finding giving reasonable suspicion of a disease or condition that would contraindicate the use of an investigational drug 2.1.2.7 Major surgery or significant traumatic injury ≤ 28 days prior to the first LMB-100 infusion
(excluding biopsies) or anticipation of the need for major surgery during study treatment 2.1.2.8 Dementia or altered mental status that would prohibit informed consent 2.1.2.9 Live attenuated vaccinations 14 days prior to treatment 2.1.2.10 Because there is an unknown but potential risk for adverse events in nursing infants secondary to treatment of the mother with LMB-100, breastfeeding should be discontinued if the mother is treated with LMB-100. These potential risks may also apply to other agents used in this study.  
2.1.2.11 Known hypersensitivity to any of the components of LMB-100 2.1.2.12 High doses of systemic corticosteroids within 7 days prior to first dosing. High dose is considered as  20 mg of dexamethasone a day (or equivalent) for  7 consecutive days.  
2.1.3 Exclusion Criteria (Arm B only) 2.1.3.1 Subjects must not have received paclitaxel or *nab-*paclitaxel within 4 months prior to initiation of study therapy 2.1.3.2 Participants with contra-indication and/or history of severe hypersensitivity reactions to nab-paclitaxel
2.2 SCREENING EVALUATION Note: Screening evaluation testing/procedures are conducted under the separate screening protocol, 01-C-0129 (Eligibility Screening and Tissue Procurement for the NIH Intramural Research Program Clinical Protocols) Screening assessments will be performed within 28 days prior to study enrollment.  
Archival tumor sample for NCI LP confirmation of diagnosis. A block of primary tissue
(or 5-10 unstained sections on charged slides) from the time of diagnosis will be required from each patient. Tissue blocks from a known recurrence will be accepted if original tumor samples are unavailable. Referring institutions will send the tumor block or 5-10 unstained sections on charged slides to CCR/NCI for correlative studies and confirmation of diagnosis. A fresh biopsy or tumor effusion sample may be collected if archival tumor tissue is not available.  
History and physical exam Vital signs including pulse oximetry ECOG performance status  
Urine or serum hCG in women of childbearing potential ECG Echocardiogram Chest X-ray
CT scan of chest, abdomen and/or pelvis and areas of known or suspected disease involvement; MRI may also be performed when appropriate FDG-PET scan CBC with differential, Acute Care Panel (sodium, potassium, chloride, bicarbonate, creatinine, glucose, BUN), Hepatic Panel (alkaline phosphatase, AST, ALT, total bilirubin, direct bilirubin), Mineral Panel (albumin, calcium, magnesium, phosphorus), creatine kinase, C-reactive protein, Coagulation (PT, PTT, fibrin degradation products)
Urinalysis Viral markers - HBSAg, anti-HCV, anti-HIV  
2.3 REGISTRATION PROCEDURES
Authorized staff must register an eligible candidate with NCI Central Registration Office (CRO) within 24 hours of signing consent. A registration Eligibility Checklist from the web site (http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) must be completed and sent via encrypted email to: NCI Central Registration Office ncicentralregistration-l@mail.nih.gov. After confirmation of eligibility at Central Registration Office, CRO staff will call pharmacy to advise them of the acceptance of the patient on the protocol prior to the release of any investigational agents. Verification of Registration will be forwarded electronically via e-mail to the research team. A recorder is available during non-working hours. 2.3.1 Treatment Assignment Procedures  
| Cohorts Letter                            | Name                                                                                              | Description   |                                                           |
|-------------------------------------------|---------------------------------------------------------------------------------------------------|---------------|-----------------------------------------------------------|
| A1                                        | Mesothelioma                                                                                      | dose          | de                                                           |
| escalation                                | Up to 18 Subjects with epithelial or biphasic mesothelioma  enrolled to determine RP2D of LMB-100 |               |                                                           |
| A2                                        | Mesothelioma                                                                                      | dose          | 7- 10 (depending on interim efficacy analysis) additional |
| expansion                                 | subjects with epithelial or biphasic mesothelioma treated at  RP2D of LMB-100                     |               |                                                           |
| B1                                        | Mesothelioma                                                                                      | with          | no                                                        |
| recent nab-paclitaxel dose  de-escalation | Up to 12 subjects with epithelial or biphasic mesothelioma  who have not been treated with nab-paclitaxel in the past 4  months enrolled to determine the RP2D of LMB-100 +nabpaclitaxel                                                                                                   |               |                                                           |  
Arms  
| B2                                    | Mesothelioma   | with   | no   |
|---------------------------------------|----------------|--------|------|
| recent nab-paclitaxel dose  expansion |                |        |      |  
| 7- 10 (depending on interim efficacy analysis) additional  subjects with epithelial or biphasic mesothelioma who have  not been treated with nab-paclitaxel in the past 4 months  treated at RP2D of LMB-100+nab-paclitaxel   |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
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arm assignment

| Arms Letter     | Name                                   | Description                                                         |
|-----------------|----------------------------------------|---------------------------------------------------------------------|
| A1              | LMB-100 dose escalation                | De-escalating doses of LMB-100 in up to 18 subjects                 |
| A2              | LMB-100 dose expansion                 | Fixed dose of LMB-100 as determined in Arm A1 in up to 16  subjects |
| B1              | LMB-100+ nab-paclitaxel                | De-escalating doses of LMB-100 + fixed dose of nabpaclitaxel in up to 12 subjects                                                                     |
| dose escalation |                                        |                                                                     |
| B2              | LMB-100+ nab-paclitaxel                | Fixed dose of LMB-100 as determined in Arm B1 + fixed dose          |
| dose expansion  | of nab-paclitaxel in up to 16 subjects |                                                                     |  
Subjects in cohort A1 are assigned to arm A1. Subjects in cohort A2 are directly assigned to arm A2. (Note: Part A of the study is now closed). Subjects in cohort B1 are assigned to arm B1. Subjects in cohort B2 are directly assigned to arm B2.
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3 study implementation 3.1 study

This is an open-label, single center Phase I study of LMB-100 in patients with advanced mesothelioma. Four arms will be assessed: Arms A1 and A2: LMB-100 monotherapy and Arms B1 and B2: LMB-100 in combination with nab-paclitaxel.  
Arms A1 and A2 (closed as of Amendment C- 03/09/17)
Up to 18 patients will be enrolled to test up to 3 de-escalating doses of LMB-100 using a standard 3+3 design (Arm A1). Initially, it was planned that up to 16 patients would be evaluated at the RP2D, now determined as 140 mcg/kg in the dose expansion portion of the study using a twostage Minimax design (Arm A2). Patients treated at the RP2D during the dose de-escalation portion of the study were included in the count of 16 patients. However, as of amendment C, 10 subjects have been treated at the RP2D and as noted in section 8, enrollment will be temporarily halted after the enrollment of 10 evaluable subjects in order to assess PK and ADA data. As no subjects have demonstrated a partial or complete response at this dose, Arms A1 and A2 will no longer be enrolling. All future subjects will be enrolled to Arms B1 and B2. Subjects previously enrolled on Arms A1 and A2 will continue to be treated as originally planned, receiving LMB-100 intravenously on days 1, 3 and 5 of each 21 day cycle. A maximum of 4 cycles of treatment will be administered.
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arms b1 and b2

Up to 12 evaluable patients will be enrolled to test up to 2 de-escalating doses of LMB-100 with a constant dose of nab-paclitaxel (Arm B1). Up to 16 patients will be evaluated at the recommended phase 2 dose (RP2D) in the dose expansion portion of the study using a two-stage Minimax design
(Arm B2). Patients treated at the RP2D during the dose de-escalation portion of the study will be included in the count of 16 patients. In both portions of Arm B, patients will receive LMB-100 intravenously on days 1, 3 and 5 of each 21 day cycle for up to 2 cycles and nab-paclitaxel on days 1 and day 8 of each 21 day cycle for up to 6 cycles.
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all arms

Pharmacokinetic measurements will be performed with each cycle. Assessment of anti-LMB-100 antibodies will be performed at the start of each cycle. Response to therapy will be assessed at baseline and then following every two cycles by CT scan. Response will be assessed by RECIST 1.1 or modified RECIST for MPM. Tumor mesothelin expression will be assessed retrospectively. Optional tumor biopsy will be performed in consenting patients when deemed feasible at the following time points: before initiation of therapy (archival tissue may also be used) and after two cycles of therapy. 3.1.1 Dose Limiting Toxicity (Dose de-escalation cohorts only) For the purpose of this study, a DLT will be defined as any of the following events attributed to LMB-100 (i.e. related to LMB-100) and occurring within 21 days after the first dose of LMB-100, the DLT period:
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hematological toxicities

Grade 4 neutropenia (i.e. absolute neutrophil count (ANC) <0.5 x 109 cells/L) for a minimum duration of 7 days)
Grade 3 and 4 febrile neutropenia (i.e. ANC < 1.0 x109 cells/L with a single temperature of >38.3°C or a sustained temperature of ≥38°C for more than one hour Grade 4 thrombocytopenia (≤25.0 x109 cells/L)
Grade 3 thrombocytopenia associated with bleeding episodes Grade ≥ 3 non-hematological toxicity with the exception of:
Alopecia (any grade) Grade 3 nausea and vomiting without appropriate treatment Grade 3 diarrhea lasting for ≤ 2 days with no fever or dehydration Infusion-related reactions **up to and including Grade 3**. They are not considered to be DLTs since, based on experience with monoclonal antibodies, IRRs are idiosyncratic and not dose-related events. Precautions will be taken if IRRs Grade ≥ 2 occur (see Section 3.3.2).  
Asymptomatic ≥ Grade 3 lymphopenia, leukopenia, hypoalbuminemia, dyselectroletemia resolving within 24 hours, increase in alkaline phosphatase.  
**Isolated** Grade 3 fever **(without signs and/or symptoms of an infection)**
occurring within 48 hours of LMB-100 infusion and resolving within 48 hours to ≤ Grade 2 and fully resolved within 1 week Grade ≥ 4 non-hematological toxicity:
Infusion-related reactions Other toxicities:  
Any other drug related toxicity considered significant enough to be qualified as a DLT in the opinion of the principal investigator.  
Inability to start cycle 2 within 3 weeks of completing cycle 1 due to drug-related adverse events.
If a patient experiences a DLT of any grade, LMB-100 will be discontinued in the patient; however, nab-paclitaxel may be continued per protocol.
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3.1.2 dose de-escalation (arm a1)

Six patients will initially be entered at dose level 1. If fewer than 2 patients experience DLT during within 21 days after the first dose of LMB-100 at the initial level, this will be the RP2D. Otherwise, dose de-escalation will proceed in cohorts of 3–6 patients. The RP2D is the dose level at which no more than 1 of up to 6 patients experience DLT during cycle 1. If a patient did not experience DLT and did not finish cycle 1, he or she will not be evaluable for toxicity and will be replaced in the dose level to ensure that at least 3 patients in each cohort have been assessed for the full DLT period prior to moving to the next dose level. If 2 or more DLTs occur at dose level -2, an amendment will be submitted in order to explore dose levels lower than 100 mcg/kg.  
| Dose De-escalation Schedule Dose Level Dose of LMB-100 in µg/kg  Level 1 170 Level -1 140 Level -2 100   |
|----------------------------------------------------------------------------------------------------------|  
Table 8: Arm A1 Dose de-escalation will follow the rules outlined below.  
| Number of Patients with  DLT    | De-escalation Decision Rule               |
|---------------------------------|-------------------------------------------|
| at Dose Level 1 0 or 1 out of 6 | This will be the recommended phase 2 dose |  
| Number of Patients with  DLT                                | De-escalation Decision Rule                                                                                                                                                                                                                                                                                      |
|-------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| at Dose Level 1 ≥ 2 out of 6                                | Enter 3 patients at the next lower dose level                                                                                                                                                                                                                                                                    |
| Number of Patients with  DLT at Any Other Given  Dose Level | De-escalation Decision Rule                                                                                                                                                                                                                                                                                      |
| 0 or 1 out of 3                                             | Enter up to 3 more patients at this dose level.  If fewer than 2 out of the 6 patients evaluated at  this dose level experience DLT, this is the  recommended phase 2 dose.   If 2 or more out of the 6 patients evaluated at this  dose level experience DLT, enter 3 patients at  the next lower dose level. |
| > 2 out of 3                                                | Enter 3 patients at the next lower dose level.                                                                                                                                                                                                                                                                   |  
3.1.3 ,Dose De-Escalation (Arm B1) Dose de-escalation will proceed in cohorts of 6 patients. Before proceeding to the next dose level, the last patient enrolled at the previous level must have completed at least one cycle of study therapy. The RP2D is the dose level at which no more than 1 of up to 6 patients experience DLT during cycle 1, and the dose below that at which at least 2 (of ≤6) patients experience DLT. If a patient did not experience DLT and did not finish treatment, he or she will not be evaluable for toxicity and will be replaced in the dose. If 2 or more DLTs occur at dose level -1, an amendment will be submitted in order to explore LMB-100 dose levels lower than 65 mcg/kg.
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table 9 arm b1 dose

| Dose De-escalation Schedule Dose Level Dose of LMB-100 in µg/kg   | Nab-paclitaxel in mg/m2   |     |
|-------------------------------------------------------------------|---------------------------|-----|
| Level -1                                                          | 65                        | 100 |
| Level 1                                                           | 100                       | 100 |  
| Number of Patients with  DLT      | De-Escalation Decision Rule                    |
|-----------------------------------|------------------------------------------------|
| at a Given Dose Level <1 out of 6 | This is the recommended phase 2 dose.          |
| > 2                               | Enter up to 6 patients at the next lower dose. |  
3.2 DRUG ADMINISTRATION
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3.2.1 lmb-100

The qualified health care professional responsible for dispensing the study drug will prepare the correct dose according to the cohort allocation of each patient. LMB-100 will be given as an IV solution on Days 1, 3, and 5 (QOD× 3) of every 21 day cycle (up to 4 cycles for Arms A1 and A2, up to 2 cycles for Arms B1 and B2). LMB-100 must be administered in a hospital or clinic equipped for IV chemotherapy. Full emergency resuscitation facilities should be immediately available and patients should be under close supervision of the investigator or delegate at all times. The compatibility and stability of the active ingredient was tested under simulated preparation/administration conditions.  
3.2.1.1 General Instructions 1. LMB-100 drug product should be inspected visually for particulates prior to administration.  
2. Do not use the solution if there is particulate matter or if it is discolored. 3. Do not shake or freeze the vial contents. 4. Ensure the drug vial content is protected from light during preparation and administration
(ambient light conditions are acceptable but avoid exposure to direct sunlight).  
5. LMB-100 drug product does not contain any preservatives. Vials are for single use only and partially used vials must not be reused.  
6. Any unused product should be kept for drug reconciliation. 7. No dilution of LMB-100 drug product into 0.9% saline bags should be performed. 8. Do not administer as IV push or bolus. 9. Other drugs that require parenteral co-administration (if applicable) should be delivered via separate infusion lines and at separate infusion sites and should not be mixed with the study drug.
3.2.1.2 Specific Instructions LMB-100 is diluted with 0.9% NaCl (1:10) **in-line** immediately prior to administration (see Figure 10 below). The undiluted LMB-100 drug product, filled in a disposable syringe, is administered by intravenous syringe infusion using a syringe driver pump. In order to allow **in-line dilution (1:10) immediately prior** to administration of neat drug product, a side flow with 0.9% NaCl must be applied (as illustrated in Figure 10). An IV infusion pump and syringe driver should be used to control the infusion rate of isotonic 0.9% NaCl solution and LMB-100 respectively. LMB-100 is administered using peripheral vein access and should not be administered using a  
 central venous catheter.  
The LMB-100 drug product should be filtered during withdrawal from the vial using a BD filter needle or alternatively, an in-line filter may be used as depicted.  
1. The syringes for administration must be prepared under appropriate aseptic conditions as LMB-100 drug product does not contain antimicrobial preservatives. All preparation should be conducted under laminar flow hood with safety glasses, lab coat and arm protection and a work procedure to minimize particle contamination.  
2. As a measure of precaution, a BD 18G (USA: 19G) blunt filter needle Versapore should be used, if no in-line filter is applied, for the withdrawal of the undiluted drug product (LMB-100) from the vial into the syringe.  
3. **Alternatively**, an in-line filter may be used to filter the undiluted drug product (LMB100) during syringe infusion before side-flow dilution. For this, an in-line filter (0.2 μm pore size) should be positioned between the syringe extension line and the injection/infusion valve (see Figure 10). The filtration must occur **before** in-line dilution with 0.9% saline.  
4. Withdraw up to 2 mL excess volume of LMB-100 to the intended dosing volume into the syringe for priming and purging the syringe extension lines before administration (see step 8).  
5. The prepared syringe should be stored and transported with a closing cone/stopper. 6. Since LMB-100 drug product does not contain antimicrobial preservatives and to comply with the chemical and physical in-use stability, the prepared syringes for infusion should be used immediately. If not used immediately, total in-use storage times of prepared syringes should not exceed 24 hours to limit microbial growth in case of potential accidental contamination. Storage conditions should generally be at 2°C to 8°C, but syringes may be held at room temperature for up to a maximum of 4 hours. The temperature during syringe preparation, storage and drug administration must not exceed 25 °C.  
7. Establish the saline flow first by flushing the lines including the 4-way stopcock and extension lines with saline.  
8. Put the filled syringe in the pre-programmed syringe driver, remove the cap and attach the infusion set. Follow the syringe driver manual/local instructions regarding how to set up and prime/purge the system before the line is attached to the closed 4-way stopcock before starting the administration.  
9. The end of infusion is defined as the time point at which the syringe driver finishes administering the total volume of LMB-100 to be infused.  
10. At the end of the first infusion, the IV line should remain in place for 2 hours. If no infusion related symptoms occur during this time, the infusion line may be removed. For subsequent infusions and if no IRR has been reported, the IV line should remain in place for at least 30 minutes from the end of the infusion. If no adverse events occur during the 30 minutes, the infusion line may be removed. If feasible, the line for drawing blood for PK samples (opposite extremity to the one with the infusion line) will remain in place until the 24-hour sample is taken.
During infusion, vital signs (including, if possible, supine diastolic and systolic blood pressure, pulse rate, and temperature) must be monitored pre-infusion, every 15 minutes (± 5 minutes) until the end of the infusion, and thereafter, every 30 minutes (± 10 minutes) until the infusion line is removed. Vital signs during the infusion are not required to be captured in the eCRF unless abnormalities are observed. LMB-100 drug product (DP) should be administered diluted using a side flow set-up at 1:10 (0.1 mg/mL DP). In order to not compromise drug product physico-chemical stability, the dilution with 0.9% NaCl should be done **in line**, immediately prior to administration of the neat DP. The infusion duration should be 30 minutes (± 5 minutes), however the duration can be increased at the discretion of the investigator based on the total dose and volume to be administered and the patient's physical condition. Syringe preparation and infusion duration should not exceed a maximum of 4 hours. In case of any adverse events related to the infusion, please refer to the specific recommendation described in section 3.3.2. 3.2.1.3 Premedications for Patients Receiving LMB-100 Due to the prevalence of infusion related reactions (IRRs) seen in the previous study of LMB-100, all patients will be predicated 30-60 minutes (+ 30 minutes) prior to each LMB-100 administration with the following medications:  
o Diphenhydramine 25-50 mg PO or IV
o Ranitidine 150 mg PO o Acetaminophen 650 mg PO
o Dexamethasone 20 mg, PO, 6-12 hours prior to LMB-100 administration, OR o Dexamethasone 10 mg, IV, 30 - 60 minutes (+30 minutes) prior to LMB-100 administration, OR
o equivalent dose of another corticosteroid as clinically indicated
(See section 3.2.2 for complete instructions on response to IRRs) Participants who experienced an IRR of Grade 3 or 4 on a previous infusion where dexamethasone or another steroid was pre-administered should not receive further LMB-100 and will be discontinued from study therapy.  
| Dose (mg)                                           | Route   |              |
|-----------------------------------------------------|---------|--------------|
| Acetaminophen                                       | 650     | Orally       |
| Ranitidine                                          | 150     | Orally       |
| Diphenhydraminea                                    | 25-50   | Orally or IV |
| Dexamethasoneb                                      | 10      | IV           |
| OR                                                  |         |              |
| Dexamethasoneb                                      | 20      | Orally       |
| a or alternative antihistamine at an adequate dose. |         |              |  
a or alternative antihistamine at an adequate dose.  
b or equivalent dose of another corticosteroid administered if a >grade 2 IRR occurred on a previous infusion despite standard pre-medication.  
3.2.2 Nab- paclitaxel (Arms B1 and B2 Only)
Nab-paclitaxel will be administered at a dose of 100 mg/m2, intravenously over 30 minutes on Days 1 and 8 of each 21 day cycle for up to 6 cycles in the absence of toxicity or disease progression.  
Detailed information about the preparation of the infusion solution and administration of nabpaclitaxel including information about compatible infusion bags, administration sets, and in line filters can be found in the package insert. 3.2.3 Co-administration of Nab-paclitaxel and LMB-100 (Arms B1 and B2 only) On days when both LMB-100 and nab-paclitaxel are both scheduled to be administered (Day 1 of cycles 1 and 2), nab-paclitaxel will be administered first. LMB-100 infusion will be initiated 30 minutes after completion of the *nab-*paclitaxel. 3.3 DOSE MODIFICATIONS  
3.3.1 For Arm A1 Subjects Treated at 170 mcg/kg only
As discussed in section 1.2.3.5, the three subjects treated at 170 mcg/kg will be subsequently dosed at 140 mcg/kg. Any further adverse events will be managed as per Table 12 below. 3.3.2 For all Subjects Table 12 below provides a guideline on how to manage certain toxicities which are expected with LMB-100 based on preclinical studies and previous clinical experience with molecules in the same class  
| Table 12 - Guidelines for Managing Specific LMB-100 Adverse Events Event Action to Be Taken IRR/hypersensitivity reaction If an IRR/hypersensitivity develops, the infusion of LMB-100  should be temporarily slowed down or interrupted. The patient  should be monitored until complete resolution of the symptoms  and treated as clinically indicated. Treatment or concomitant  medication may include acetaminophen, antihistamine, IV  saline, oxygen, bronchodilators, corticosteroids, and  vasopressors depending on the symptoms. If the infusion is interrupted: o In the event of IRR CTCAE Grade1, upon resolution of  symptoms, the infusion will resume at the same rate (the  rate being used at the time that the IRR occurred). o In the event of IRR Grade 2 or 3, upon resolution of  symptoms, the infusion will resume at one-half the  previous rate. The infusion can be re-escalated to initial  rate if considered well tolerated after 1 hour of infusion. o In the event of IRR CTCAE Grade 3, or CTCAE Grade 4  (which may include pulmonary or cardiac events) or an  anaphylactic reaction:  The infusion must be stopped and the patient should  receive aggressive treatment   |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| Version Date: 02/16/2021 Event                                       | Action to Be Taken o Patients experiencing                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | IRR   | CTCAE   | Grade   | 4   | or   |
|----------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------|---------|---------|-----|------|
| anaphylaxis must be permanently discontinued from  LMB-100 treatment |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |       |         |         |     |      |
| Vascular leak syndrome                                               | In the event of Grade ≥2 CTCAE vascular leak syndrome  (medical intervention indicated): o Delay LMB-100 administration until complete resolution  of the event o For hypotension minimize fluid resuscitation to avoid  fluid overload Minimize crystalloid solutions (e.g., saline) o Vasopressor support (e.g., phenylephrine) if indicated to  stabilize blood pressure o Administer colloidal solutions (e.g., albumin) if there is a  clinically significant and persistent systolic blood  pressure drop, and the patient is symptomatic, or urine  output declines o For pulmonary congestion provide diuretic and/or  albumin treatment in case of hypoalbuminemia as  appropriate o Progressive shortness of breath may require in addition  endotracheal intubation or drainage of a pleural effusion o For oliguria and /or rising serum creatinine level delay  LMB-100 if Grade C3 urine output ( <10 mL/hr) o Use fluids judiciously if increase in urine output is  required o Use dopamine if patient is unresponsive to or unable to  tolerate fluids Monitor serum albumin levels prior to each  LMB-100 treatment cycle o In the event of Grade ≥2 CTCAE pericardial effusion  (asymptomatic effusion small to moderate size), consider  delaying LMB-100 administration. In the event of Grade  ≥3 CTCAE pericardial effusion (effusion with  physiologic consequences) stop LMB-100 treatment until  full resolution o Hydrocortisone (200 mg IV) or equivalent dose of another |       |         |         |     |      |
| Inflammatory                                                         | reactions                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             | to    |         |         |     |      |
| serosal membranes                                                    | corticosteroid as clinically indicated                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |       |         |         |     |      |
| o                                                                    | In the event of Grade 2 CTCAE pericardial effusion  (asymptomatic effusion small to moderate size), consider  delaying LMB-100 administration. In the event of Grade  ≥3 CTCAE pericardial effusion (effusion with                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |       |         |         |     |      |  
| Event                                                                                      | Action to Be Taken physiologic consequences) stop LMB-100 treatment until  full resolution o In the event of pleuritis resulting in mild to severe pleuritic  pain, treat with analgesics or steroids as clinically  indicated o For patients who have previously experienced pleuritis  consider administration of a tapering course of prednisone  for 7 days starting with the next LMB-100 infusion   |
|--------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Renal Toxicity                                                                             | Periodic monitoring of renal function and serum electrolytes IV fluids 0.9 NaCl may be administered after LMB-100  infusion if clinically indicated because of increasing serum  creatinine or decreased urine output.                                                                                                                                                                                    |
| IRR = infusion related reaction; IV = intravenous; CTCAE = Common Terminology Criteria for |                                                                                                                                                                                                                                                                                                                                                                                                           |  
IRR = infusion related reaction; IV = intravenous; CTCAE = Common Terminology Criteria for Adverse Events  
3.3.3 Nab-paclitaxel If nab-paclitaxel is withdrawn prior to cycle 2, LMB-100 may still be continued in the absence of progressive disease. Dose modifications of *nab-*paclitaxel for myelosuppression and other chemotherapy related toxicities will be made as per the NSCLC instructions in the package insert. 3.4 STUDY CALENDAR 3.4.1 Arms A1 and A2 Calendar 1 cycle =21 days Screening assessments must occur within 28 days prior to enrollment. If screening assessments are performed within 3 days prior to dosing, the assessments do not need to be repeated on C1D1 unless otherwise indicated (see ECG and pregnancy test below). Assessments after C1D1 may be performed up to 3 days prior to indicated time unless otherwise indicated.  
Dosing cycles after cycle 1 may be delayed for up to two weeks to accommodate schedule conflicts, Federal holidays and inclement weather, etc.  
| weather, etc.                                | Cycle 1   | Subsequent Cycles   | Post Therapy Follow-up   |     |     |     |     |     |          |           |           |         |           |
|----------------------------------------------|-----------|---------------------|--------------------------|-----|-----|-----|-----|-----|----------|-----------|-----------|---------|-----------|
| Day                                          | Day       | Day                 | Day                      | Day | Day | Day | Day | Day | Day      | Follow-Up | Long-Term |         |           |
| Procedure                                    | Screening | 1                   | 3                        | 5   | 8   | 15  | 1   | 3   | 5        | 8         | 15        | Visit13 | Follow Up |
| LMB-100                                      | X         | X                   | X                        | X   | X   | X   |     |     |          |           |           |         |           |
| History and PE                               | X         | X                   | X                        | X   | X   | X   | X   | X   | X        | C2  only  | X         |         |           |
| Weight                                       | X         | X                   | X                        | X   | X   | X   | X   | X   | C2  only | X         |           |         |           |
| Height                                       | X         |                     |                          |     |     |     |     |     |          |           |           |         |           |
| Vital signs1                                 | X         | X                   | X                        | X   | X   | X   | X   | X   | X        | C2  only  | X         |         |           |
| Performance Score                            | X         | X                   | X                        |     |     |     |     |     |          |           |           |         |           |
| Labs2                                        | X         | X                   | X                        | X   | X   | X   | X   | X   | X        | X         | X         |         |           |
| Urinalysis                                   | X         | X                   | X                        |     |     |     |     |     |          |           |           |         |           |
| HLA Typing (Class I  and Class II)           | X3        |                     |                          |     |     |     |     |     |          |           |           |         |           |
| Anti HIV, anti HCV,  HBSAg                   | X         |                     |                          |     |     |     |     |     |          |           |           |         |           |
| Urine or serum hCG in  women of childbearing | X         | X5                  | X                        | X   |     |     |     |     |          |           |           |         |           |
| potential4 Confirmation of dx6               | X         |                     |                          |     |     |     |     |     |          |           |           |         |           |
| NIH                                          | Advance   |                     |                          |     |     |     |     |     |          |           |           |         |           |
| Directives Form                              | X7        |                     |                          |     |     |     |     |     |          |           |           |         |           |  
| Cycle 1                                    | Subsequent Cycles                                                                                                                           | Post Therapy Follow-up   |     |     |     |     |     |     |     |           |           |         |           |
|--------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------|--------------------------|-----|-----|-----|-----|-----|-----|-----|-----------|-----------|---------|-----------|
| Day                                        | Day                                                                                                                                         | Day                      | Day | Day | Day | Day | Day | Day | Day | Follow-Up | Long-Term |         |           |
| Procedure                                  | Screening                                                                                                                                   | 1                        | 3   | 5   | 8   | 15  | 1   | 3   | 5   | 8         | 15        | Visit13 | Follow Up |
| Biopsy (optional)                          | X8                                                                                                                                          | C3  only9                |     |     |     |     |     |     |     |           |           |         |           |
| Correlative                                | Research                                                                                                                                    |                          |     |     |     |     |     |     |     |           |           |         |           |
| Studies                                    | Please see section 5.2                                                                                                                      |                          |     |     |     |     |     |     |     |           |           |         |           |
| PK                                         | Please see section 5.2.1                                                                                                                    |                          |     |     |     |     |     |     |     |           |           |         |           |
| CT CAP and/or if  clinically indicated     | X                                                                                                                                           | Every 6 weeks ± 7 days   | X10 | X14 |     |     |     |     |     |           |           |         |           |
| MRI +/- gadolinium FDG-PET                 | X                                                                                                                                           | Every 6 weeks ± 7 days   | X10 | X14 |     |     |     |     |     |           |           |         |           |
| ECG11                                      | X                                                                                                                                           | X                        | X   | X   | X   | X   | X   | X   |     |           |           |         |           |
| Echocardiogram                             | X                                                                                                                                           |                          |     |     |     |     |     |     |     |           |           |         |           |
| Chest X-Ray12                              | X                                                                                                                                           | X                        | X   |     |     |     |     |     |     |           |           |         |           |
| Annual phone call to  monitor survival and | X                                                                                                                                           |                          |     |     |     |     |     |     |     |           |           |         |           |
| additional CA therapy Adverse Events       | Monitored continuously                                                                                                                      |                          |     |     |     |     |     |     |     |           |           |         |           |
| Concomitant  Medications                   | Monitored continuously                                                                                                                      |                          |     |     |     |     |     |     |     |           |           |         |           |
| 1                                          | At screening: heart rate, blood pressure, body temperature, pulse oximetry. During the infusions of all study medication in Cycles 1 and 2, |                          |     |     |     |     |     |     |     |           |           |         |           |  
Adverse Events Monitored continuously Concomitant Medications Monitored continuously 1 **At screening:** heart rate, blood pressure, body temperature, pulse oximetry. **During the infusions** of all study medication in Cycles 1 and 2, vital signs (heart rate, blood pressure, body temperature) have to be monitored pre-infusion and every 15 minutes (± 5 minutes) during the infusion and then every 30 minutes (± 10 minutes) from the end of the infusion until the infusion line is removed. From Cycle 3 onwards, vital signs have to be monitored only pre and post infusion if study medication has been tolerated well in previous cycles.  
2 CBC with differential, Acute Care Panel, Hepatic Panel, Mineral Panel, creatine kinase, C-reactive protein, PT, PTT, fibrin degradation products. 3 May be performed after study consent is signed but prior to treatment initiation (baseline)
4 Required in women of childbearing potential; i.e. premenopausal women and women ≤ 2 years after menopause (menopause is defined as amenorrhea for > 2 years.  
5 Only required if more than 14 days have passed since screening. 6 Please see section 2.2 for tissue requirements 7 As indicated in section 12.3, all subjects will be offered the opportunity to complete an NIH advance directives form. This should be done preferably at baseline but can be done at any time during the study as long as the capacity to do so is retained. The completion of the form is strongly recommended, but is not required.  
8 To be performed at baseline - any time after patient has signed the study consent, but before study therapy initiation. 9 To be performed after the completion of 2 cycles of study therapy.  
10 Performed only if patient removed from study therapy for reason other than progressive disease 11 Single 12-lead ECG will be recorded at screening (within 7 days before first dose of study treatment), then pre- and end of LMB-100 infusion for first 3 cycles and at the withdrawal and follow-up visit. Pre-infusion at all other study drug administrations. Additional unscheduled ECG assessments should be performed if cardiovascular symptoms or abnormalities occur.  
12 Chest X-rays will be obtained at screening and pre-infusion time on indicated dosing days. In addition, Chest X-ray will be performed when clinically indicated during the study treatment period. Chest X-rays will be obtained at the time of the tumor assessments, when convenient 13 The assessments listed refer to those that will be performed if the patient is seen in clinic 4- 6 weeks after the last dose of study drug. If the patient is unable to return to the clinic for the follow up visit, an adverse event assessment will be performed by telephone.  
14 Scans performed only in patients who have not had progressive disease. Scans will continue every 6 weeks until disease progression.  
3.4.2 Arms B1 and B2 Calendar
untitled h1
1 cycle =21 days

Screening assessments must occur within 28 days prior to enrollment. If screening assessments are performed within 3 days prior to dosing, the assessments do not need to be repeated on C1D1 unless otherwise indicated (see ECG and pregnancy test below). Assessments after C1D1 may be performed up to 3 days prior to indicated time unless otherwise indicated. Dosing cycles after cycle 1 may be delayed for up to two weeks to accommodate schedule conflicts, Federal holidays and inclement weather, etc.  
| Cycle 1 and cycle 2                                           | Subsequent Cycles   | Post Therapy Follow-up   |     |     |          |          |     |     |     |           |           |         |           |
|---------------------------------------------------------------|---------------------|--------------------------|-----|-----|----------|----------|-----|-----|-----|-----------|-----------|---------|-----------|
| Day                                                           | Day                 | Day                      | Day | Day | Day      | Day      | Day | Day | Day | Follow-Up | Long-Term |         |           |
| Procedure                                                     | Screening           | 1                        | 3   | 5   | 8        | 15       | 1   | 3   | 5   | 8         | 15        | Visit13 | Follow Up |
| LMB-100                                                       | X                   | X                        | X   |     |          |          |     |     |     |           |           |         |           |
| Nab-paclitaxel                                                | X                   | X                        | X   | X   |          |          |     |     |     |           |           |         |           |
| History and PE                                                | X                   | X                        | X   | X   | X        | C1  only | X   | X   | X   |           |           |         |           |
| Weight                                                        | X                   | X                        | X   | X   | C1  only | X        | X   | X   |     |           |           |         |           |
| Height                                                        | X                   |                          |     |     |          |          |     |     |     |           |           |         |           |
| Vital signs1                                                  | X                   | X                        | X   | X   | X        | C1  only | X   | X   | X   |           |           |         |           |
| Performance Score                                             | X                   | X                        | X   |     |          |          |     |     |     |           |           |         |           |
| Labs2                                                         | X                   | X                        | X   | X   | X        | C1  only | X   | X   | X   |           |           |         |           |
| Urinalysis                                                    | X                   | X                        | X   |     |          |          |     |     |     |           |           |         |           |
| C1  only  3                                                   |                     |                          |     |     |          |          |     |     |     |           |           |         |           |
| HLA Typing (Class I  and Class II) Anti HIV, anti HCV,  HBSAg | X                   |                          |     |     |          |          |     |     |     |           |           |         |           |
| Urine or serum hCG in  women of childbearing                  | X                   | X5                       | X   | X   |          |          |     |     |     |           |           |         |           |
| potential4 Confirmation of dx6                                | X                   |                          |     |     |          |          |     |     |     |           |           |         |           |
| NIH                                                           | Advance             |                          |     |     |          |          |     |     |     |           |           |         |           |
| Directives Form                                               | X7                  |                          |     |     |          |          |     |     |     |           |           |         |           |  
| Cycle 1 and cycle 2                        | Subsequent Cycles                                                                                                                           | Post Therapy Follow-up   |     |     |     |     |     |     |     |           |           |         |           |
|--------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------|--------------------------|-----|-----|-----|-----|-----|-----|-----|-----------|-----------|---------|-----------|
| Day                                        | Day                                                                                                                                         | Day                      | Day | Day | Day | Day | Day | Day | Day | Follow-Up | Long-Term |         |           |
| Procedure                                  | Screening                                                                                                                                   | 1                        | 3   | 5   | 8   | 15  | 1   | 3   | 5   | 8         | 15        | Visit13 | Follow Up |
| Biopsy (optional)                          | X8                                                                                                                                          | C3  only9                |     |     |     |     |     |     |     |           |           |         |           |
| Correlative                                | Research                                                                                                                                    |                          |     |     |     |     |     |     |     |           |           |         |           |
| Studies                                    | Please see section 5.2.2                                                                                                                    |                          |     |     |     |     |     |     |     |           |           |         |           |
| PK                                         | Please see section 5.2.2                                                                                                                    |                          |     |     |     |     |     |     |     |           |           |         |           |
| CT CAP and/or if  clinically indicated     | X                                                                                                                                           | Every 6 weeks ± 7 days   | X10 | X14 |     |     |     |     |     |           |           |         |           |
| MRI +/- gadolinium FDG-PET                 | X                                                                                                                                           | Every 6 weeks ± 7 days   | X10 | X14 |     |     |     |     |     |           |           |         |           |
| ECG11                                      | X                                                                                                                                           | X                        | X   | X   | X   | X   |     |     |     |           |           |         |           |
| Echocardiogram                             | X                                                                                                                                           |                          |     |     |     |     |     |     |     |           |           |         |           |
| Chest X-Ray12                              | X                                                                                                                                           | C2                       | C1  |     |     |     |     |     |     |           |           |         |           |
| only                                       | only                                                                                                                                        |                          |     |     |     |     |     |     |     |           |           |         |           |
| Annual phone call to  monitor survival and | X                                                                                                                                           |                          |     |     |     |     |     |     |     |           |           |         |           |
| additional CA therapy Adverse Events       | Monitored continuously                                                                                                                      |                          |     |     |     |     |     |     |     |           |           |         |           |
| Concomitant  Medications                   | Monitored continuously                                                                                                                      |                          |     |     |     |     |     |     |     |           |           |         |           |
| 1                                          | At screening: heart rate, blood pressure, body temperature, pulse oximetry. During the infusions of all study medication in Cycles 1 and 2, |                          |     |     |     |     |     |     |     |           |           |         |           |  
Adverse Events Monitored continuously Concomitant  
Medications Monitored continuously  
1 **At screening:** heart rate, blood pressure, body temperature, pulse oximetry. **During the infusions** of all study medication in Cycles 1 and 2,  
vital signs (heart rate, blood pressure, body temperature) have to be monitored pre-infusion and every 15 minutes (± 5 minutes) during the infusion and then every 30 minutes (± 10 minutes) from the end of the infusion until the infusion line is removed. From Cycle 3 onwards, vital signs have to be monitored only pre and post infusion if study medication has been tolerated well in previous cycles.  
2 CBC with differential, Acute Care Panel, Hepatic Panel, Mineral Panel, creatine kinase, C-reactive protein, PT, PTT, fibrin degradation products.  
3 May be performed after study consent is signed but prior to treatment initiation (baseline) 4 Required in women of childbearing potential; i.e. premenopausal women and women ≤ 2 years after menopause (menopause is defined as  
amenorrhea for > 2 years.  
5 Only required if more than 14 days have passed since screening.  
6 Please see section 2.2 for tissue requirements 7 As indicated in section 12.3, all subjects will be offered the opportunity to complete an NIH advance directives form. This should be done  
preferably at baseline but can be done at any time during the study as long as the capacity to do so is retained. The completion of the form is  
strongly recommended, but is not required.  
8 To be performed at baseline - any time after patient has signed the study consent, but before study therapy initiation.  
9 To be performed after the completion of 2 cycles of study therapy.  
10 Performed only if patient removed from study therapy for reason other than progressive disease 11 Single 12-lead ECG will be recorded at screening (within 7 days before first dose of study treatment), then pre- and end of LMB-100 infusion for the first 2 cycles and at the withdrawal and follow-up visit. Pre-infusion at all other study drug administrations. Additional unscheduled ECG assessments should be performed if cardiovascular symptoms or abnormalities occur.  
12 Chest X-rays will be obtained at screening and pre-infusion time on indicated dosing days. In addition, Chest X-ray will be performed when clinically indicated during the study treatment period. Chest X-rays will be obtained at the time of the tumor assessments, when convenient 13 The assessments listed refer to those that will be performed if the patient is seen in clinic 4- 6 weeks after the last dose of study therapy. If the patient is unable to return to the clinic for the follow up visit, an adverse event assessment will be performed by telephone.  
14 Scans performed only in patients who have not had progressive disease. Scans will continue every 6 weeks until disease progression.  
3.5 CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF STUDY CRITERIA  
3.5.1 Criteria for removal from protocol therapy
Prior to removal from study, a safety follow up visit as indicated in section 3.5.2 must be completed.  
Progressive disease  
Arm A1 or A2 participant has completed 4 cycles of study therapy Arm B1 or B2 participant has completed 6 cycles of study therapy Participant requests to be withdrawn from active therapy Pregnancy Unacceptable Toxicity as defined in sections 3.1.1 and 3.3 Investigator discretion 3.5.2 Follow Up
All patients will be asked to return for a follow up visit ~ 4 - 6 weeks after the last dose of the study drug (see Study Calendar for assessments). If the subject is unable to return to the NIH, a brief assessment for adverse events will be performed by telephone. Subjects who have not had progressive disease will continue to have follow-up scans every 6 weeks until disease progression. Thereafter, all patients or their physicians will be contacted annually by telephone to assess survival status and additional anti-cancer therapy. 3.5.3 Off-Study Criteria  
Patient lost to follow-up Participant requests to be withdrawn from study+ Death
+ Patient will be required to complete follow up visit prior to removal from study therapy for this reason.  
3.5.4 Off Protocol Therapy and Off Study Procedure Authorized staff must notify Central Registration Office (CRO) when a subject is taken off protocol therapy and when a subject is taken off-study. A Participant Status Updates Form from the web site (http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) main page must be completed and sent via encrypted email to: NCI Central Registration Office ncicentralregistration-l@mail.nih.gov.
untitled h1
4 concomitant medications/measures 4.1.1 permitted

Concomitant therapy includes any medication (e.g., prescription drugs, over-the-counter (OTC) drugs, approved dietary and herbal supplements, nutritional supplements) used by a patient from the screening period until the follow-up visit. If any treatment is given within 4 weeks prior to screening this should be reported to the investigator and recorded in the eCRF. All therapy and/or medication administered to manage adverse events should be recorded on the Adverse Event eCRF
untitled h1
4.1.2 prohibited therapy

Patients should be treated for all concomitant conditions and adverse events according to accepted standards of medical care at the discretion of the investigator. The following treatments are not permitted during the study:
Any other investigational therapy  
Cytotoxic chemotherapy agents other than study agents Radiotherapy. Note: palliative 8Gy radiotherapy is allowed.  
Immunosuppressive therapy and chronically administered glucocorticoids (high dose is considered as  20 mg of dexamethasone a day [or equivalent)] for  7 consecutive days)
Other systemic anti-neoplastic agents and targeted therapies
If any anti-neoplastic or investigational therapies listed above are needed, the patient will be considered to have evidence of progressive neoplastic disease and have experienced treatment failure with study treatment and should be withdrawn from study treatment. All concomitant treatments must be documented in the eCRF.
untitled h1
5 biospecimen collection

5.1 CORRELATIVE STUDIES FOR RESEARCH/PHARMACOKINETIC STUDIES  
5.1.1 Pharmacokinetic Assessments
All blood samples for PK assessment will be collected from an IV line different to that receiving the infusion to measure free and total concentrations for LMB-100 for all patients. The date and time of each sample collection will be recorded. If multiple samples are drawn at a given time point, the PK sample should take precedence. Free and total plasma concentrations of LMB-100 will be measured using validated ligand-binding assays. 5.1.1.1 Sample collection:
Blood will be collected in 2 mL K2EDTA tubes (purple top) at the times defined in section 5.2.  
Samples should be inverted 8 to 10 times after collection. Store on wet ice or at 4°C. Processing within 60 minutes of blood collection is highly preferred.
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5.1.1.2 sample processing

Samples will be processed in the Clinical Pharmacology Program. Please e-mail NCIBloodcore@mail.nih.gov at least 24 hours before transporting samples (the Friday before is preferred). For sample pickup, page 102-11964. For immediate help, call 240-760-6180 (main blood processing core number) or, if no answer, 240-760-6190 (main clinical pharmacology lab number). For questions regarding sample processing, contact NCIBloodcore@mail.nih.gov. Upon arrival in the CPP the following procedures should be followed:  
1. Store on wet ice until centrifugation. 2. Centrifuge 1500xg for 10 minutes at 4°C within 60 minutes of blood collection. 3. Transfer plasma specimen to 2mL cryovials and store at -70°C.
The analyses will be performed retrospectively in batched samples or at the end of the trial. 5.1.1.3 Sample Shipping Samples will be shipped by the CPP on dry ice to Dr. Jon Inglefield in Frederick for analysis.  
Leidos Biomedical, Inc. Attention: Ms. Yanyu Wang, Dr. Jon Inglefield Building 469, Room 120 Miller Drive Frederick, MD 21702 Phone: 301-846-6905/301-846-6865 5.1.1.4 Sample storage Samples will be stored in the CPP until shipment to the Leidos Biomedical Inc. Lab in Frederick. 5.1.2 Assessment of anti-drug antibodies (ADAs) 5.1.2.1 Sample Collection Samples will be collected before first dose of LMB-100 administration during each cycle in which LMB-100 is administered (See section 5.2)
Draw 2mL into K2EDTA tube (purple top). Samples should be inverted 8 to 10 times after collection. Store on wet ice or at 4°C. Processing within 60 minutes of blood collection is highly preferred. 5.1.2.2 Sample Processing Samples will be processed in the Clinical Pharmacology Program. Please e-mail NCIBloodcore@mail.nih.gov at least 24 hours before transporting samples (the Friday before is preferred). For sample pickup, page 102-11964. For immediate help, call 240-760-6180 (main blood processing core number) or, if no answer, 240-760-6190 (main clinical pharmacology lab number). For questions regarding sample processing, contact NCIBloodcore@mail.nih.gov. Upon arrival in the CPP, each sample should be processed in the following manner:  
1. Store on wet ice until centrifugation.  
2. Centrifuge 1500xg for 10 minutes at 4°C within 60 minutes of blood collection. 3. Transfer plasma specimen to 2mL cryovials and store at -70°C.
Autoantibody levels will be retrospectively assessed. 5.1.2.3 Sample Shipping Samples will be shipped by the CPP on dry ice to Dr. Jon Inglefield in Frederick for analysis.  
Leidos Biomedical, Inc. Attention: Ms. Yanyu Wang, Dr. Jon Inglefield Building 469, Room 120 Miller Drive Frederick, MD 21702 Phone: 301-846-6905/301-846-6865 5.1.2.4 Sample Storage Samples will be stored in the CPP until shipment to the Leidos Biomedical Inc. Lab in Frederick. 5.1.3 Mesothelin and Megakaryocyte Potentiating Factor (MPF) Serum Samples: The levels of serum mesothelin as well as megakaryocyte potentiating factor, which is released into serum from the processing of mesothelin precursor protein will be assessed in order to determine correlation with therapeutic response. 5.1.3.1 Sample Collection Samples will be obtained as follows:
Arms A1 and A2 - prior to the first LMB-100 dose of each cycle and at the end of treatment  
Arms B1 and B2 -prior to the first LMB-100 dose of cycles 1 and 2, prior to the first nabpaclitaxel administration during cycles 3 - 6, and at the end of treatment.
All blood samples will be taken by either direct venipuncture or an indwelling venous access. At each sample collection time, blood (2mL) will be drawn into a 3.5-mL serum separator tube (tiger top tube) labeled as follows:
- Subject ID Number
- Study Number - Time and date of collection 5.1.3.2 Sample Processing Please e-mail NCIBloodcore@mail.nih.gov at least 24 hours before transporting samples (the Friday before is preferred). For sample pickup, page 102-11964. For immediate help, call 240-760-6180 (main blood processing core number) or, if no answer, 240-760-6190 (main clinical pharmacology lab number). For questions regarding sample processing, contact NCIBloodcore@mail.nih.gov. Upon arrival in the CPP, each sample should be processed in the following manner: Allow blood to clot for 10 minutes and centrifuge to separate the serum within 30 minutes of collection. If unable to process within 30 minutes, then whole blood tubes may be stored upright in refrigerator (4-8C) for up to 48 hours prior to processing. Processing of samples within 30 minutes is strongly preferred. Stability studies will establish if degradation of soluble mesothelin in whole blood during 0.5 to 48 hours is significant and therefore if the data from these samples should be included in the analysis. Transfer the serum into two pre-labeled cryotubes and immediately freeze by placing on dry ice.  
Transfer frozen serum samples into a - 80C freezer for storage.  
5.1.3.3 Sample Storage All serum samples will be stored by Dr. Figg's Clinical Pharmacology Program. 5.1.4 Retrospective Analysis of Mesothelin Expression in tumor tissue IHC analysis will be performed by the Laboratory of Pathology at NCI to determine mesothelin expression within the tumor at any time after study enrollment. Leftover tissue from archival specimens or tumor biopsies obtained at screening or from optional collections at baseline or after cycle 2 may be used for this purpose. Specimens will be used to correlate treatment with response with mesothelin expression in an exploratory analysis.
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5.1.4.1 specimen collection

Collection of optional tumor biopsies should be guided by ultrasound, CT scan, or other method according to the location of the selected lesion using a ≤ 18 gauge needle to provide cores ideally of at least 20 mm in length or equivalent size. At least 2, ideally 4 core biopsies will be obtained at each time point (baseline and after cycle 2). Fine needle aspiration and biopsy of bone lesions are not acceptable. All biopsies collected under this protocol will undergo review in the NCI Laboratory of Pathology.
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5.1.5 cytokines for identification of

PE-based RITs cause dose-limiting CLS. At low doses CLS manifests as mild and transient weight gain, hypoalbuminemia, and peripheral or facial edema. At higher doses it can cause lifethreatening cardiopulmonary compromise. Previous studies in rats have indicated that pathological changes indicative of CLS onset occur within just two hours of toxin administration and even when the PE fragment lacks a targeting domain.[32] In vitro studies with cultured endothelial cells have demonstrated that super-physiologic doses of PE-based RITs cannot induce endothelial cell toxicity unless the cells express the RIT target.[33] Together these data suggest the hypothesis that PE-based RITs cause CLS by triggering release of vasoactive cytokines by specific immune cells rather than through direct damage to endothelial cells.  
5.1.5.1 Specimen collection To test this hypothesis, we will collect additional serum from participants during each of LMB100 therapy on:
Days 1, 3, and 5 (Pre-dose)
Day 8 in 4 mL red SST tubes 5.1.5.2 Sample processing Please e-mail NCIBloodcore@mail.nih.gov at least 24 hours before transporting samples (the Friday before is preferred). For sample pickup, page 102-11964. For immediate help, call 240-760-6180 (main blood processing core number) or, if no answer, 240-760-6190 (main clinical pharmacology lab number). For questions regarding sample processing, contact NCIBloodcore@mail.nih.gov. Cytokine levels will be retrospectively assessed. 5.1.6 Gene expression-based characterization of the immune landscape before and after treatment with LMB-100 As referenced above, optional tumor biopsy will be performed in consenting patients when deemed feasible at the following time points: before initiation of therapy (archival tissue may also be used) and after two cycles of therapy. We will evaluate tumor biopsies before and after treatment with LMB-100 (using samples referenced in section above) using a hybridization-based digital gene expression platform nCounter (NanoString Technologies). This platform allows for unbiased multiplexed quantification of RNA transcripts achieving sensitivity comparable to quantitative reverse-transcription polymerase chain reaction (Q-RT-PCR), without any enzymatic reaction involved in the process. We have expertise in the use of this technology for the characterization of purified cell subsets and also for the study of the changes that occur in human tumors, as a consequence of a given treatment, using tumor core biopsy tissues. This analysis can be performed on flash frozen biopsies without any further isolation or enrichment of specific cell types. For the present study, we will screen pre- and post-treatment samples for the expression of markers of immune cell subsets (CD3, CD8, CD4, etc.), local production of cytokines (interferon-gamma, tumor necrosis factor-alpha, etc.) and chemokines (CXCL13, CCL5, etc.), adhesion molecules and others. The purpose of this study is to characterize the molecular changes that occur within the tumors following treatment with LMB-100. The ultimate goal is to gain a better understanding of the mechanism of action of this treatment, and to identify molecular correlates of clinical outcomes such as objective responses and/or improved survival. In order to achieve these goals, flash frozen samples will be subjected to total RNA isolation followed by hybridization with capture and detection probes specific for 620 transcripts, including genes involved in the regulation of the immune function and also markers expressed by tumor cells and tumor stroma. To cover those target transcripts, a combination of a commercially available pre-designed probe set (GX Human Immunology v2, NanoString Technologies) will be used in combination with a custom-designed code set of thirty additional targets (Panel Plus, NanoString Technologies). Hybridization complexes will be quantified using a NanoString nCounter Analysis System, at the Genomics Core Facility of the Center for Cancer Research, NCI. Results obtained by this approach will be correlated with data obtained from immunohistochemistry of tumor biopsies as well as with data from analysis of peripheral blood populations, for a comprehensive study of the mechanism of action of LMB-100 in mesothelioma patients. Samples will be stored in the Laboratory of Dr. Raffit Hassan, Building 10, Room 3B51. 5.1.7 Mapping T cell epitopes in LMB-100. Identification and investigation of T cell epitopes in therapeutic proteins is important for understanding of the immunogenicity response against LMB-100. Identification of those T cell epitopes will allow us to engineer the next generation of immunotoxin which will be less immunogenic because it will have less T cell epitopes. Method: For T cell epitope mapping we isolate PBMC from patients that were previously treated with the immunotoxin and had neutralizing antibodies. PBMC are frozen at a concentration of 1.5 x107 cells/ml/vial. Cells are viably frozen in the following freezing media: RPMI, 10% human AB
serum, 1% P/S (antibiotic) and 7.5% DMSO. The PBMC are stimulated with the whole protein and the activated T cells are expanded using human IL2 which is a lymphocyte stimulator. Next, the in vitro expanded PBMC are re- stimulated with a peptide library spanning the sequence of the Fab portion of the immunotoxin. The T cell activation is detected by monitoring secretion of IL2 using IL2 ELISpot. An illustration of the method, the controls and threshold determination were previously described in Mazor et al 2012.[20] 5.1.7.1 Sample collection Samples will be collected at baseline and at the end of treatment in eight 8 mL whole blood tubes (black and blue)." [BD Vacutainer® CPT (REF 362761)] 5.1.7.2 Sample processing Please e-mail NCIBloodcore@mail.nih.gov at least 24 hours before transporting samples (the Friday before is preferred). For sample pickup, page 102-11964. For immediate help, call 240-760-6180 (main blood processing core number) or, if no answer, 240-760-6190 (main clinical pharmacology lab number).  
For questions regarding sample processing, contact NCIBloodcore@mail.nih.gov. PBMCs will be processed within 6 hours post-collection using Ficoll- Hypaque density-gradient separation according to manufacturer's instructions. Collect the buffy coat and wash three times with Dulbecco's PBS without Ca and Mg. Cryopreserve in liquid nitrogen at a concentration of 1.5 x 107 cells/mL in RPMI1640 supplemented with 5% human AB serum and 7.5% DMSO.  
5.1.7.3 Sample storage Samples will be stored in by the Clinical Pharmacology Program; analyses will be performed in the laboratory of Dr. Raffit Hassan.  
5.1.8 Assessment of Myeloid Derived Suppressor Cells (MDSC) in Subjects with Mesothelioma 5.1.8.1 Sample collection
Samples will be collected at baseline prior to first dose of LMB-100 and the end of cycle 2 and at the end of treatment. All blood samples will be taken by either direct venipuncture or an indwelling venous access. At each sample collection time, blood (30 mL) will be drawn into three 10 mL sodium heparin (green top) tubes. The tube should be labeled with the following information:
- Subject ID Number - Study Number - Time and date of collection -Specimen type: **peripheral blood**
5.1.8.2 Sample processing The samples will be sent to NIH flow lab located at 3S240, South part of Building 10.  
All specimens for flow cytometry will be scheduled in advance by contacting Dr. StetlerStevenson stetler@mail.nih.gov and/or Dr. Yuan yuanc@mail.nih.gov by email. Peripheral blood samples should arrive no later than 1 pm to the flow cytometry laboratory on the day scheduled. Specimens are received in the laboratory via escort or via delivery by someone from the clinical team. Specimens for delivery AFTER HOURS ONLY may also be placed in the secure specimen drop box located in Laboratory of Pathology 3N hallway. Peripheral blood specimens are collected in sodium heparin tubes, and are stored at room temperature until delivery. The specimens should be delivered as soon as possible after collection, and cannot be evaluated if > 48 hours old. Upon receipt in the flow lab, the sample will be checked for correct labeling with appropriate patient identifiers and specimen type (e.g. peripheral blood).