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Version Date: *02/16/21* NIH Protocol \#: 18C0136 Version Date: February 16, 2021 NCT Number: NCT03644550 Title: Phase II Study of the Anti-Mesothelin Immunotoxin LMB-100 Followed by Pembrolizumab in Malignant Mesothelioma NCI Principal Investigator: Raffit Hassan, M.D.  
Thoracic and GI Malignancies Branch (TGMB), CCR, NCI Building 10, Room 4-5330 9000 Rockville Pike Bethesda, MD 20892 Telephone: 240-760-6232 E-mail: hassanr@mail.nih.gov
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investigational agents

| Drug Name:    | LMB-100                    | Pembrolizumab (Keytruda®)   |
|---------------|----------------------------|-----------------------------|
| IND Number:   | 152907                     | 152907                      |
| Sponsor:      | Center for Cancer Research | Center for Cancer Research  |
| Manufacturer: | Selecta Biosciences        | Merck                       |  
Commercial Agents: None 1
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precis

Background:
LMB-100, and a closely related immunotoxin, SS1P, also targeting mesothelin, have been studied in previous Phase 1 clinical studies for mesothelioma and pancreatic cancer.  
LMB-100 has demonstrated anti-tumor efficacy against several mesothelin expressing tumor models including mesothelioma PDX models PD-1 (encoded by the gene *Pdcd1*) is an Ig superfamily member related to CD28 and CTLA-4 that has been shown to negatively regulate antigen receptor signaling upon engagement of its ligands The PD-1 receptor-ligand interaction is a major pathway hijacked by tumors to suppress immune control. The normal function of PD-1, expressed on the cell surface of activated T-cells under healthy conditions, is to down-modulate unwanted or excessive immune responses, including autoimmune reactions.  
In immune-competent mice bearing human mesothelin expressing tumors local administration of LMB-100 with CTLA-4 blockade eradicates murine tumors by promoting anti-cancer immunity.  
LMB-100 treatment increase CD8+ T cell infiltration in murine lung adenocarcinoma tumors that express human mesothelin.  
Combination treatment with LMB-100 plus anti-PD1 results in greater anti-tumor efficacy in murine lung cancer model Pembrolizumab is an anti PD-1 antibody that has demonstrated responses of long duration in clinical trials and has generally been well-tolerated It is hypothesized that the anti-mesothelin immunotoxin LMB-100 followed by pembrolizumab will result in greater anti-tumor efficacy in patients with mesothelioma.  
Objectives:
To determine the objective response rate of sequential therapy with LMB-100 followed by pembrolizumab in subjects with pleural and peritoneal mesothelioma.  
Eligibility:
Histologically confirmed epithelial or biphasic pleural or peritoneal mesothelioma (with
<50% sarcomatoid component) not amenable to potentially curative surgical resection.  
Subjects must have at least one prior chemotherapy regimen that includes pemetrexed and cisplatin or carboplatin.  
Age ≥ 18 years.  
ECOG performance status of 0 or 1. Adequate organ and bone marrow function Prior PD1/PD-L1 inhibitor treatment is prohibited Chemotherapy within 4 weeks or radiotherapy within 2 weeks prior to start of study therapy is prohibited.  
2 Subjects with active CNS metastasis are excluded Subjects with active autoimmune disease for which they had received systemic treatment during the previous 2 years receiving systemic glucocorticoids (excluding daily glucocorticoid-replacement therapy for conditions such as adrenal or pituitary insufficiency) are excluded Subjects with active interstitial lung disease, or a history of pneumonitis for which they had received glucocorticoids are excluded Design:
This is an open-label, single center phase II study of LMB-100 followed by pembrolizumab in subjects with advanced pleural or peritoneal mesothelioma who have progressed on standard therapies.  
Subjects will receive LMB-100 at the single agent MTD on days 1, 3 and 5 of a 21-day cycle for 2 cycles and pembrolizumab 200 mg on day1 of each subsequent 21-day cycle until disease progression (on or after pembrolizumab) or intolerable toxicity for a maximum of 2 years (unless second course initiated).  
Tumor biopsies will be performed at baseline, at the end of cycle 2 and at the end of cycle 4 to evaluate changes in the tumor immune microenvironment following treatment with LMB-100 and pembrolizumab.  
Up to 35 evaluable subjects will be enrolled  
| TABLE OF CONTENTS                                                                                                                                        |                                                                                                                                                      |                                                                                                                                              |
|----------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------|
| PRÉCIS                                                                                                                                                   |                                                                                                                                                      |                                                                                                                                              |
| TABLE OF CONTENTS ..............................................................................................................................4        |                                                                                                                                                      |                                                                                                                                              |
| STATEMENT OF COMPLIANCE ................................................................................................................10               |                                                                                                                                                      |                                                                                                                                              |
| INTRODUCTION                                                                                                                                             |                                                                                                                                                      |                                                                                                                                              |
| 1                                                                                                                                                        | 1.1                                                                                                                                                  | Study Objectives .........................................................................................................................10 |
| 1.1.1                                                                                                                                                    | Primary Objective .......................................................................................................................10          |                                                                                                                                              |
| 1.1.2                                                                                                                                                    | Secondary Objectives                                                                                                                                 |                                                                                                                                              |
| 1.1.3                                                                                                                                                    | Exploratory Objectives                                                                                                                               |                                                                                                                                              |
| 1.2                                                                                                                                                      | Background and Rationale ..............................................................................................................11            |                                                                                                                                              |
| 1.2.1                                                                                                                                                    | Hypotheses .                                                                                                                                         |                                                                                                                                              |
| Background on Mesothelioma ..........................................................................................................11                  |                                                                                                                                                      |                                                                                                                                              |
| 1.2.2                                                                                                                                                    |                                                                                                                                                      |                                                                                                                                              |
| 1.2.3                                                                                                                                                    | Mesothelin as a target for cancer therapy                                                                                                            |                                                                                                                                              |
| 1.2.4                                                                                                                                                    | LMB-100:..........................................................................................................................................12 |                                                                                                                                              |
| 1.2.5                                                                                                                                                    | Pembrolizumab                                                                                                                                        |                                                                                                                                              |
| 1.2.6                                                                                                                                                    | Rationale for Combination Therapy........................................................................................23                          |                                                                                                                                              |
| Justification for Study Doses ....................................................................................................................25     |                                                                                                                                                      |                                                                                                                                              |
| 1.2.7                                                                                                                                                    |                                                                                                                                                      |                                                                                                                                              |
| 2                                                                                                                                                        | ELIGIBILITY ASSESSMENT AND ENROLLMENT ................................................................................27                             |                                                                                                                                              |
| Eligibility Criteria .................................................................................................................................27 |                                                                                                                                                      |                                                                                                                                              |
| 2.1                                                                                                                                                      | 2.1.1                                                                                                                                                | Inclusion Criteria                                                                                                                           |
| 2.1.2                                                                                                                                                    | Exclusion Criteria                                                                                                                                   |                                                                                                                                              |
| 2.1.3                                                                                                                                                    | Recruitment Strategies ...............................................................................................................31             |                                                                                                                                              |
| 2.2                                                                                                                                                      | Screening Evaluation                                                                                                                                 |                                                                                                                                              |
| 2.2.1                                                                                                                                                    | Screening activities performed prior to obtaining informed consent ..........................31                                                      |                                                                                                                                              |
| 2.2.2                                                                                                                                                    | Screening activities performed after a consent for screening has been signed .........31                                                             |                                                                                                                                              |
| 2.3                                                                                                                                                      | Participant Registration and Status Update Procedures ..................................................32                                           |                                                                                                                                              |
| Treatment Assignment Procedures                                                                                                                          |                                                                                                                                                      |                                                                                                                                              |
| 2.3.1                                                                                                                                                    |                                                                                                                                                      |                                                                                                                                              |
| 3                                                                                                                                                        | STUDY IMPLEMENTATION                                                                                                                                 |                                                                                                                                              |
| 3.1                                                                                                                                                      | Study Design .                                                                                                                                       |                                                                                                                                              |
| SCHEMA .                                                                                                                                                 |                                                                                                                                                      |                                                                                                                                              |
| 3.1.1                                                                                                                                                    |                                                                                                                                                      |                                                                                                                                              |
| 3.1.2                                                                                                                                                    | Second Course                                                                                                                                        |                                                                                                                                              |
| 3.2                                                                                                                                                      | D Drug Administration ...................................................................................................................34          |                                                                                                                                              |  
4  
Version Date: *02/16/21*  
| Version Date: 02/16/21 3.2.1 LMB-100 34 3.2.2 Pembrolizumab 37 3.3 Dose Modifications/Delays 37 3.3.1 LMB-100 37 3.3.2 Pembrolizumab 40 3.4 Study Calendar 46 3.5 Criteria for Removal from Protocol Therapy and Off Study Criteria 50 3.5.1 Criteria for removal from protocol therapy 50 3.5.2 Off-Study Criteria 50 3.6 Clinical Criteria for Early Trial Termination 50 4 CONCOMITANT MEDICATIONS/MEASURES 51 4.1 Permitted Therapy 51 4.2 Prohibited Therapy 51 5 BIOSPECIMEN COLLECTION 52 5.1 Correlative Studies for Research/Pharmacokinetic Studies 52 5.1.1 Pharmacokinetic Assessments 52 5.1.2 Assessment of anti-drug antibodies (ADAs) 53 5.1.3 Retrospective Analysis of Mesothelin and PD-L1 Expression in tumor tissue 54 5.1.4 Mesothelin and Megakaryocyte Potentiating Factor (MPF) Serum Samples 54 5.1.5 Gene expression-based characterization of the immune landscape before and after  treatment with LMB-100 and pembrolizumab 55 5.1.6 Tumor Microenvironment Studies Using Multiplex Staining Technologies 56 5.1.7 Retrospective analysis of MSI status 56 5.1.8 Mechanism of Capillary Leak Syndrome (CLS) 56 5.2 Sample Collection Schedule 58 5.3 Sample Storage, Tracking and Disposition 60 5.3.1 Clinical Pharmacology Program 60 5.3.2 Leidos Biomedical, Inc. Lab 60 5.3.3 NCI Laboratory of Pathology 61 5.3.4 Laboratory of Dr. Raffit Hassan 61 5.3.5 CAT-I Laboratory 61 5.3.6 Protocol Completion/Sample Destruction 62   |
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abbreviated title lmb-100 plus pembrolizumab

| Version Date:  02/16/21                                                                                                                                      |                                                                                                                                                                 |                 |
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| DATA COLLECTION AND EVALUATION ....................................................................................62                                        |                                                                                                                                                                 |                 |
| 6                                                                                                                                                            | 6.1                                                                                                                                                             | DATA COLLECTION |
| 6.2                                                                                                                                                          | DATA Sharing Plans ............................................................................................................................63               |                 |
| Human Data Sharing Plan ........................................................................................................63                           |                                                                                                                                                                 |                 |
| 6.2.1                                                                                                                                                        |                                                                                                                                                                 |                 |
| 6.2.2                                                                                                                                                        | Genomic Data Sharing Plan .......................................................................................................63                             |                 |
| 6.3                                                                                                                                                          | Response Criteria ............................................................................................................................63                |                 |
| 6.3.1                                                                                                                                                        | Peritoneal Mesothelioma                                                                                                                                         |                 |
| Pleural Mesothelioma                                                                                                                                         |                                                                                                                                                                 |                 |
| 6.3.2                                                                                                                                                        |                                                                                                                                                                 |                 |
| 6.3.3                                                                                                                                                        | iRECIST ..............................................................................................................................................70        |                 |
| 6.3.4                                                                                                                                                        | D Duration of Response ......................................................................................................................................74 |                 |
| Progression-Free Survival ..............................................................................................................74                   |                                                                                                                                                                 |                 |
| 6.3.5                                                                                                                                                        |                                                                                                                                                                 |                 |
| 6.3.6                                                                                                                                                        | Objective Response Rate..............................................................................................................74                         |                 |
| 6.4                                                                                                                                                          | Toxicity Criteria ..................................................................................................................................74          |                 |
| 7                                                                                                                                                            | NIH REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN .....74                                                                                              |                 |
| Definitions                                                                                                                                                  |                                                                                                                                                                 |                 |
| 7.1                                                                                                                                                          |                                                                                                                                                                 |                 |
| 7.2                                                                                                                                                          | OHSRP Office of Compliance and Training / IRB Reporting ..........................................74                                                            |                 |
| 7.2.1                                                                                                                                                        | Expedited Reporting                                                                                                                                             |                 |
| 7.2.2                                                                                                                                                        | IRB Requirements for PI Reporting at Continuing Review ...............................................74                                                        |                 |
| 7.3                                                                                                                                                          | NCI Clinical Director Reporting                                                                                                                                 |                 |
| 7.4                                                                                                                                                          | DATA AND SAFETY MONITORING Plan                                                                                                                                 |                 |
| 7.4.1                                                                                                                                                        | Principal Investigator/Research Team ....................................................................................75                                     |                 |
| Se                                                                                                                                                           |                                                                                                                                                                 |                 |
| 7.4.2                                                                                                                                                        |                                                                                                                                                                 |                 |
| SPONSOR REPORTING CRITERIA ..........................................................................................................75                      |                                                                                                                                                                 |                 |
| 8                                                                                                                                                            | 8.1                                                                                                                                                             | Definitions     |
| A Adverse Event ..........................................................................................................................................75 |                                                                                                                                                                 |                 |
| 8.1.1                                                                                                                                                        | Serious Adverse Event (SAE)                                                                                                                                     |                 |
| 8.1.2                                                                                                                                                        |                                                                                                                                                                 |                 |
| 8.1.3                                                                                                                                                        | Events to Be Treated as Adverse Events for Manufacturer Reporting ........................76                                                                    |                 |
| 8.1.4                                                                                                                                                        | Life-threatening                                                                                                                                                |                 |
| Severity                                                                                                                                                     |                                                                                                                                                                 |                 |
| 8.1.5                                                                                                                                                        |                                                                                                                                                                 |                 |
| 8.1.6                                                                                                                                                        | Relationship to Study Product..........................................................................................................77                       |                 |
| OVERDOSE                                                                                                                                                     |                                                                                                                                                                 |                 |
| 8.1.7                                                                                                                                                        |                                                                                                                                                                 |                 |
| 8.1.8                                                                                                                                                        | Events of clinical interest ..................................................................................................................77                |                 |  
6 Abbreviated Title: LMB-100 plus pembrolizumab  
| Version Date: 02/16/21                                                                                                                           |                                                                                                                                                 |
|--------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------|
| Assessment of Safety Events                                                                                                                      |                                                                                                                                                 |
| 8.2                                                                                                                                              |                                                                                                                                                 |
| 8.3                                                                                                                                              | Reporting of Serious Adverse Events ......................................................................................78                    |
| 8.4                                                                                                                                              | Safety Reporting Criteria to the Pharmaceutical Collaborators ......................................78                                          |
| Reported by Sponsor to Manufacturer ....................................................................................78                       |                                                                                                                                                 |
| 8.4.1                                                                                                                                            |                                                                                                                                                 |
| 8.4.2                                                                                                                                            | Reported by Study Team to Manufacturer                                                                                                          |
| 8.5                                                                                                                                              | Reporting Pregnancy .......................................................................................................................81   |
| 8.5.1                                                                                                                                            | Maternal exposure                                                                                                                               |
| Paternal exposure ............................................................................................................................81 |                                                                                                                                                 |
| 8.5.2                                                                                                                                            |                                                                                                                                                 |
| 8.6                                                                                                                                              | Regulatory Reporting for Studies Conducted Under CCR-Sponsored IND..............81                                                              |
| 9                                                                                                                                                | CLINICAL MONITORING PLAN ..............................................................................................................81       |
| 10 STATISTICAL CONSIDERATIONS .................................................................................................82                |                                                                                                                                                 |
| Statistical Hypothesis .......................................................................................................................82 |                                                                                                                                                 |
| 10.1                                                                                                                                             |                                                                                                                                                 |
| 10.2                                                                                                                                             | Sample Size Determination ..........................................................................................................82          |
| 10.3                                                                                                                                             | Populations for Analyses                                                                                                                        |
| 10.3.1 Evaluable for toxicity ................................................................................................................83 |                                                                                                                                                 |
| 10.3.2 Evaluable for objective response: .........................................................................................83             |                                                                                                                                                 |
| 10.4                                                                                                                                             | Statistical Analyses ........................................................................................................................83 |
| 10.4.1 General Approach .................................................................................................................83      |                                                                                                                                                 |
| 10.4.2  Analysis of the Primary Endpoints                                                                                                        |                                                                                                                                                 |
| 10.4.3  Analysis of the Secondary Endpoints                                                                                                      |                                                                                                                                                 |
| 10.4.4 Safety Analyses                                                                                                                           |                                                                                                                                                 |
| 10.4.5  Baseline Descriptive Statistics ............................................................................................84           |                                                                                                                                                 |
| 10.4.6 Planned Interim Analyses                                                                                                                  |                                                                                                                                                 |
| 10.4.7 Sub-Group Analyses ...............................................................................................................84      |                                                                                                                                                 |
| 10.4.8 Tabulation of individual Participant Data ...........................................................................84                   |                                                                                                                                                 |
| 10.4.9  Exploratory Analyses ...............................................................................................................84   |                                                                                                                                                 |
| 11                                                                                                                                               | COLLABORATIVE AGREEMENTS .................................................................................................84                    |
| 1.1                                                                                                                                              | Clinical Trials Agreement (CTA) ............................................................................................84                  |
| 12 HUMAN SUBJECTS PROTECTIONS                                                                                                                    |                                                                                                                                                 |
| 12.1                                                                                                                                             | Rationale For Subject Selection .................................................................................................84             |
| 12.2                                                                                                                                             | Participation of Children ..........................................................................................................85          |
| 12.3                                                                                                                                             | Participation of Subjects Unable to Give Consent ................................................................85                             |  
7  
Version Date: *02/16/21*  
| Version Date: 02/16/21 12.4 Evaluation of Benefits and Risks/Discomforts 85 12.4.1 Risks from Study Drugs 85 12.4.2 Blood Collection 85 12.4.3 CT Scans 85 12.4.4 Image Guided Biopsy Collection 85 12.4.5 Radiation 85 12.5 Risks/Benefits Analysis 86 12.6 Consent Process and Documentation 86 12.6.1 Request for Waiver of Consent for Screening Activities 87 13 REGULATORY AND OPERATIONAL CONSIDERATIONS 87 13.1 Quality Assurance and Quality Control 87 13.2 Conflict of Interest Policy 87 13.3 Confidentiality and Privacy 87 14 PHARMACEUTICAL INFORMATION 88 14.1 LMB-100 (IND # 152907) 88 14.1.1 Source 88 14.1.2 Toxicity 88 14.1.3 Handling 90 14.1.4 Formulation and preparation 90 14.1.5 Stability and Storage 90 14.1.6 Administration procedures 91 14.1.7 Incompatibilities 91 14.2 Pembrolizumab 93 14.2.1 Source 93 14.2.2 Clinical Supplies Disclosure 93 14.2.3 Toxicity 93 14.2.4 Formulation and preparation 108 14.2.5 Stability and Storage 108 14.2.6 Administration procedures 108 14.2.7 Incompatibilities 108 14.2.8 Returns and Reconciliation 109 15 REFERENCES 110   |
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| 16   | APPENDICES 114                                                                                                 |
|------|----------------------------------------------------------------------------------------------------------------|
| 16.1 | Appendix A: Performance Status Criteria 114                                                                    |
| 16.2 | Appendix B: Contraception Requirements 115                                                                     |
| 16.3 | Appendix C: Imaging and Treatment after First Radiologic Evidence of Progressive  Disease on Pembrolizumab 117 |
| 16.4 | Appendix D: Second Course (Retreatment) Imaging 118                                                            |  
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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 determine the objective response rate of sequential therapy with LMB-100 followed by pembrolizumab in subjects with pleural and peritoneal mesothelioma.  
1.1.2 Secondary Objectives Safety of sequential administration of pembrolizumab following LMB-100 To determine the duration of response, progression free survival and overall survival.  
1.1.3 Exploratory Objectives To establish the correlation of response with tumor mesothelin expression. To evaluate correlation of tumor response with tumor PD-L1 expression.  
To evaluate changes in the tumor microenvironment following treatment with LMB-100 and pembrolizumab.  
To evaluate the utility of serum mesothelin and megakaryocyte potentiating factor as a biomarkers of tumor response.  
To define the pharmacokinetics characteristics of LMB-100 in order to correlate responses with LMB-100 blood levels To determine the incidence of antibody development at the end of cycles 1 and 2 with this combination To evaluate MSI status in participants demonstrating objective response To determine the mechanism of capillary leak syndrome (CLS)
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1.2 background and rationale 1.2.1

Anti-mesothelin immunotoxin LMB-100 followed by pembrolizumab will result in greater anti-tumor efficacy in subjects with mesothelioma by augmenting anti-tumor immunity.  
LMB-100 will induce tumor inflammation that leads to recruitment of CD8+ cells in the tumor and administration of pembrolizumab will increase the efficacy of these cytotoxic T cells.
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1.2.2 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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1.2.3 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, peritoneum and pericardium.[8] 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).[9] 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.[10]
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.[11] 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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1.2.4 lmb-100

Recombinant immunotoxins are therapeutic agents composed of an antibody fragment attached to a protein toxin derived from bacteria or plants. After entering the cell by endocytosis, the immunotoxin reaches the cytosol, where it inactivates elongation factor 2, arrests protein synthesis and induces apoptotic cell death. LMB-100 is a recombinant immunotoxin consisting of a humanized anti-mesothelin Fab fused to a 24kDa truncated *Pseudomonas* exotoxin A (PE)
fragment with mutations that suppress B and T cell epitopes.[12] It has anti-tumor efficacy against several mesothelin expressing tumor models including mesothelioma PDX models.[13] Phase I
clinical trial of LMB-100 has been completed at NCI and we have established the MTD, which will be used in combination studies with pembrolizumab.
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1.2.4.1 rationale for the development

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 1 for structure) is a next generation PE-fusion protein that has been protein-engineered to maximally reduce its immunogenicity by:  
1. Using a fully humanized Fab fragment derived from the anti-mesothelin antibody SS1 for tumor targeting
2. 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  
3. 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,[14] but has also resulted in reduced incidents of CLS in animal models of CLS.[15]
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1.2.4.2 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 1). 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.[15, 16] 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.
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1.2.4.3 nonclinical studies 1.2.4.3.1 nonclinical

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 mesothelin-positive 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.[12, 17]
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 LMB-100 in monotherapy or in combination with standard chemotherapies may provide clinical benefit to patients with cancer.
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1.2.4.3.2 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 LMB100 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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1.2.4.3.3 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 CLS. 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.
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1.2.4.4 clinical testing of single

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 MSLN-expressing 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 years and 53.3% were female. All participants had received prior anticancer 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 pre-medications 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 capillary 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.  
| Table 1. LMB-100 Dose escalation study- NCT02317419                |                 |              |
|--------------------------------------------------------------------|-----------------|--------------|
| 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 capillary leak syndrome and proteinuria                  |                 |              |
| NE, Study terminated before DLT assessment period was complete and |                 |              |  
NE, Study terminated before DLT assessment period was complete and  
  
  
patients only received single dose of LMB-100
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1.2.4.4.1 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 capillary leak syndrome, pyrexia, atrial flutter/fibrillation, 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. 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 CLS which manifested with hypotension, respiratory compromise, serosal membrane reaction and hyponatremia as well as the hypoalbuminemia and edema that can be seen with mild CLS. Other symptoms associated with the DLT dose were fatigue, nausea, vomiting, decreased appetite and mild elevation of transaminases.  
1.2.4.5 Clinical testing of single agent LMB-100 at NCI (Study 16C0127 -ongoing)
As of September 24, 2018, twenty-one patients were enrolled on the study. Ten patients were treated with single agent LMB-100; 11 with a combination of LMB-100 plus nab-paclitaxel. Of these 21 patients 12 had peritoneal mesothelioma and 9 pleural mesothelioma; 12 female and 9 male. 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). Since increase in serum creatinine was a common toxicity pattern at this dose level it was defined as DLT per protocol established criteria. The protocol was subsequently amended 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 of the protocol accordingly. All subsequent patients have been treated at 140 mcg/kg. Of the 21 patients; 9 patients were taken off treatment due to disease progression; 8 completed the study with stable disease. Out of the 21 patients who received treatment on this study, 9 are still alive. The single agent MTD of LMB-100 was established as 140 mcg/kg given on days 1, 3 and 5 of a 21-day cycle.  
Grade 2 infusion reactions were seen in 6 patients at some point during the treatment with LMB100. In 3 patients it was seen during cycle 2, and in the remaining patients t during cycle 3 or cycle 4 . 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. In one patient under Arm B who received 2 cycles of LMB-100 and 6 cycles of nab-paclitaxel, LMB-100 was held for cycle 2 day 5 after a reaction in cycle 2 day 3. The most frequently occurring events were hypoalbuminemia (22% of 278 events) anemia (14%) lymphocyte count decreased (13%), hypophosphatemia (5%)Most of the events were grade 2. There was one serious grade 3 thromboembolic event; however, it was not attributed to the IND.
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1.2.4.5.1 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; 30-min 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 for the 10 patients treated with single agent LMB-100 is shown in Table 2 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 2, all 10 patients had good LMB-100 blood levels during cycle 1. However, only 5 of 10 patients had good blood levels during cycle 2. None of the 8 patients who got cycle 3 and 4 of LMB-100 had good LMB-100 blood levels during cycle 3 or 4.  
| Table 2. Decrease in LMB-100 Exposure Over Time 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   |
| 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     | 140     | BQL  |
| 5                                                                                                                                                     | 140     | 1124    | 140     | BQL     | 140     | ‒       | 140     | ‒    |
| 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     | 1526    | 140     | 27.8    | 140     | 20      | 140     | BQL  |
| 10                                                                                                                                                    | 140     | 2689    | 140     | 5.1     | 140     | N/A     | 140     | N/A  |
| * BQL: below assay's quantifiable limit (2.1 ng/mL); ‒ Because of disease progression patient did not receive cycle  3 and 4; N/A: data not available |         |         |         |         |         |         |         |      |  
Note: Anti-drug (LMB-100) antibodies (ADAs) were measured pre-dose on C1D1. Inhibition percentages 41.8%
indicate presence of pre-existing ADAs These results show that all patients can have good blood levels during cycle 1 and half had detectable blood levels during cycle 2. 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.
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1.2.5 pembrolizumab

Pembrolizumab is a potent humanized immunoglobulin G4 (IgG4) monoclonal antibody (mAb) with high specificity of binding to the programmed cell death 1 (PD-1) receptor, thus inhibiting its interaction with programmed cell death ligand 1 (PD-L1) and programmed cell death ligand 2 (PD-L2). Based on preclinical in vitro data, pembrolizumab has high affinity and potent receptor blocking activity for PD-1. Pembrolizumab has an acceptable preclinical safety profile and is in clinical development as an intravenous (IV) immunotherapy for advanced malignancies.  
Keytruda® (pembrolizumab) is indicated for the treatment of patients across a number of malignancies because of its mechanism of action to bind the PD-1 receptor on the T cell.
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1.2.5.1 pharmaceutical and therapeutic information

The importance of intact immune surveillance function in controlling outgrowth of neoplastic transformations has been known for decades.[18] Accumulating evidence shows a correlation between tumor-infiltrating lymphocytes in cancer tissue and favorable prognosis in various malignancies. In particular, the presence of CD8+ T-cells and the ratio of CD8+ effector T-cells/FoxP3+ regulatory T-cells (T-regs) correlates with improved prognosis and long-term survival in solid malignancies, such as ovarian, colorectal, and pancreatic cancer; hepatocellular carcinoma; malignant melanoma; and renal cell carcinoma. Tumor-infiltrating lymphocytes can be expanded ex vivo and reinfused, inducing durable objective tumor responses in cancers such as melanoma.[19, 20]
The PD-1 receptor-ligand interaction is a major pathway hijacked by tumors to suppress immune control. The normal function of PD-1, expressed on the cell surface of activated T-cells under healthy conditions, is to down-modulate unwanted or excessive immune responses, including autoimmune reactions. PD-1 (encoded by the gene *Pdcd1*) is an immunoglobulin (Ig)
superfamily member related to cluster of differentiation 28 (CD28) and cytotoxic T-lymphocyteassociated protein 4 (CTLA-4) that has been shown to negatively regulate antigen receptor signaling upon engagement of its ligands (PD-L1 and/or PD-L2).[21, 22]
The structure of murine PD-1 has been resolved.[23] PD-1 and its family members are type I
transmembrane glycoproteins containing an Ig-variable–type (IgV-type) domain responsible for ligand binding and a cytoplasmic tail responsible for the binding of signaling molecules. The cytoplasmic tail of PD-1 contains 2 tyrosine-based signaling motifs, an immunoreceptor tyrosine-based inhibition motif, and an immunoreceptor tyrosine-based switch motif. Following T-cell stimulation, PD-1 recruits the tyrosine phosphatases, SHP-1 and SHP-2, to the immunoreceptor tyrosine-based switch motif within its cytoplasmic tail, leading to the dephosphorylation of effector molecules such as CD3 zeta (CD3ζ), protein kinase C-theta (PKCθ), and zeta-chain-associated protein kinase (ZAP70), which are involved in the CD3 T-cell signaling cascade.[22, 24-26] The mechanism by which PD-1 down-modulates T-cell responses is similar to, but distinct from, that of CTLA-4, because both molecules regulate an overlapping set of signaling proteins.[27, 28] As a consequence, the PD-1/PD-L1 pathway is an attractive target for therapeutic intervention in mesothelioma.
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1.2.5.2 preclinical and clinical trial

The nonclinical toxicity studies consisted of pivotal 1-month and a 6-month repeat-dose chronic toxicity studies with 4-month recovery periods in Cynomolgus monkeys. These studies were supported by toxicokinetic evaluation of pembrolizumab. Additional evaluation included 2 in vitro tissue cross-reactivity studies with pembrolizumab in normal human and Cynomolgus monkey tissues, respectively, and immunotoxicology testing using surrogate antimurine PD-1 mAb in a TDAR study in mice to address whether treatment with anti-PD-1 would result in potential immune-mediated toxicity following vaccination and recall responses. There were no findings of toxicological significance in any of the conducted studies.
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pharmacology

No traditional metabolism studies were conducted with pembrolizumab per current ICH S6 (R1) guidance on preclinical safety evaluation of biotechnology-derived pharmaceuticals. However, in vivo studies were conducted in C.B17 SCID mice to demonstrate the lack of Fab-arm or half molecule exchange for pembrolizumab. IgG4 wild-type molecules can undergo in vitro and in vivo molecular rearrangement called Fab-arm (or half molecule) exchange by swapping their half molecule with other IgG4 half molecules, thereby generating bispecific or hybrid antibodies.[29, 30] A point mutation (S228P) in the core hinge region in IgG4 has been shown to be sufficient to prevent the Fab-arm exchange.[29, 30] The results supported that pembrolizumab, which has a hinge mutation from S to P at position 228, did not form detectable hybrid antibodies with co-administered wild type IgG4 molecules in vivo in SCID mice (PK007). This observation is consistent with the results of extensive in vitro characterization of pembrolizumab (PK007) and indicates that pembrolizumab is not likely to engage in Fab-arm exchange in humans.
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assessment of effects of pd-1

Antimouse PD-1 J43 was tested as a monotherapy in the MC38 (colon adenocarcinoma in C57Bl/6 mice), C1498 (acute myeloid leukemia in C57Bl/6 mice), PDV6 (squamous cell carcinoma in C57Bl/6 mice), and A20 (B cell lymphoma in BALB/c mice) syngeneic mouse tumor models. In all monotherapy experiments, tumor cells were implanted subcutaneously in syngeneic hosts and were staged at 50 to 80 mm3 before dosing was initiated. Antimouse PD-1 or isotype control antibody was administered intraperitoneally every 3 to 4 days for a total of 5 treatments. Efficacy was determined by monitoring tumor volumes and long-term survival for each experimental group. PD-1 blockade demonstrated antitumor efficacy in each of these syngeneic tumor models. Anti-PD-1 therapy also enhanced the effect of chemotherapeutic agents such as gemcitabine and 5-FU with combination therapy resulting in increased efficacy and increased complete regression rates in vivo.  
In this study, MC38 colon adenocarcinomas were staged to approximately 100 to 120 mm3 before initiation of concurrent treatment with 5-FU (40 mg/kg) and antimouse PD-1 (10 mg/kg), both administered intraperitoneally once every 3 days for a total of 5 injections. The combined treatment of MC38 colon adenocarcinomas with 5-FU and antimouse PD-1 showed a significant increase in antitumor efficacy over the individual monotherapy groups. This increased efficacy was reflected in a 60% complete regression rate in the combined treatment protocol. In the monotherapy groups, anti–PD-1 alone induced 20% (2 out of 10) complete responses, whereas none of the mice treated with control antibody or 5-FU plus control antibody demonstrated complete regression. PD-1 blockade using the antimouse PD-1 J43 surrogate antibody in combination with gemcitabine also showed enhanced efficacy in the mouse MC38 colon adenocarcinoma tumor model. 1.2.5.2.2 Clinical Investigations
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pharmacokinetics

Pembrolizumab PK samples have been obtained from multiple trials for various tumor types including melanoma, NSCLC, HNSCC, UC and microsatellite instability high (MSI-H) tumors. The doses tested in these tumor types include one or more of the following doses: 2 mg/kg Q3W,
10 mg/kg Q3W, 10 mg/kg every 2 weeks (Q2W), and 200 mg Q3W.  
The PK profile of pembrolizumab is consistent with that of other humanized mAbs, which typically have a low CL and a limited central volume of distribution (Vc).[31-33] The estimates of between subject variability are low-to-moderate and are within the range of historically reported variability levels for mAbs. A recent review of mAbs reported the between subject variability range of 15% to 65% for CL and a median (range) of 26% (12%– 84%) for Vc.[31]
Steady-state concentrations of pembrolizumab are reached by 16 weeks of repeated dosing with a Q3W regimen and the systemic accumulation is 2.1-fold. The peak concentration, trough concentration, and area under the plasma concentration versus time curve at steady state of pembrolizumab increased dose proportionally in the dose range of 2 to 10 mg/kg Q3W. As pembrolizumab is an IgG4 antibody that is administered parentally and cleared by catabolism, food and drug-drug interactions (DDI) are not anticipated to affect exposure. Therefore, no dedicated DDI studies have been performed. However, as systemic corticosteroids may be used to treat immune-mediated adverse reactions concomitant with pembrolizumab, the potential for a PK DDI with pembrolizumab as a victim was assessed. No relationship was observed between prolonged use of systemic corticosteroids and pembrolizumab exposure.
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safety

Pembrolizumab has been granted approval in a number of markets and indications and has an established safety profile. The overall safety profile of pembrolizumab is derived primarily from a locked and verified dataset with pooled data from monotherapy clinical trials (n-2799) in melanoma and NSCLC, as part of the product development of pembrolizumab. In addition, the manufacturer continues to analyze data from other ongoing and completed clinical trials, as well as from the post marketing environment. The mechanism of action of pembrolizumab involves the interruption of the binding of PD-1 to its ligands, thereby interrupting the down-modulation of T-cell immune response. It is therefore anticipated that ARs associated with pembrolizumab would include immune-mediated AEs. Based upon the mechanism of action, Merck developed a broad list of immune-mediated adverse events of special interest (AEOSI) to evaluate and monitor. Additionally, though not immunemediated, infusion-related reactions are also included in the AEOSI list. Based on ongoing monitoring, Merck has identified those AEOSIs that have been observed and assessed as related to pembrolizumab. These events form the basis of the current safety profile for the product. In addition, several AEOSIs (encephalitis, myocarditis, myasthenic syndrome, sarcoidosis, as well as the further characterization of severe skin reactions to include fatal Stevens Johnson Syndrome [SJS] and toxic epidermal necrolysis [TEN]) have been identified from data sources primarily outside the verified dataset. The majority of participants, 2727 or 97.4%, experienced 1 or more AEs, and 2062 (73.7%) experienced 1 or more AEs reported as drug-related by the investigator. The percentage of participants who experienced SAEs was lower; 1042 (37.2%) of participants experienced 1 or more SAEs; 334 (11.9%) participants discontinued due to an AE, and 282 (10.1%) participants experienced a drug-related SAE, as determined by the investigator. The 5 most frequently reported AEs were: fatigue (37.3%), nausea (24.5%), decreased appetite (22.5%), diarrhea (22.3%), and cough (22%). The 5 most frequently reported SAEs were pneumonia (3.0%), pleural effusion (1.7%), pneumonitis (1.6%), dyspnea (1.6%) and pulmonary embolism (1.5%). The 5 most frequently reported AEs considered drug related by the investigator were fatigue (24.2%), pruritus (16.7%), rash (13.8%), diarrhea (12.3%), and nausea (10.9%). The 5 most frequently reported SAEs considered drug-related by the investigator were pneumonitis (1.6%), colitis (0.9%), diarrhea (0.6%), pyrexia (0.4%), and autoimmune hepatitis (0.3%).  
The core risk profile of pembrolizumab was updated in IB v. 15 based on post-marketing data through 31-MAR-2017 to add a new potential risk of graft versus host disease (GVHD) after pembrolizumab in patients with a history of allogeneic hematopoietic stem cell transplant
(HSCT). A further update was made based on data through 03-SEP-2018 to add VogtKoyanagi-Harada syndrome and hemophagocytic lymphohistiocytosis as adverse drug reactions.  
Pembrolizumab has a positive benefit-risk profile and is well tolerated in the approved indications, as evidenced by a low rate of toxicity Grade 3 to 5 drug-related AEs (13.8%), discontinuations due to AEs (11.9%), and deaths due to drug-related AEs (0.4%). Furthermore, the frequency of immune-mediated AEOSIs is low, and these events are readily managed in the clinical setting.
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efficacy

Pembrolizumab monotherapy and combination therapies have been administered to participants with hematologic malignancies and solid tumors in Merck-sponsored trials. Approved indications include: melanoma, NSCLC, HNSCC, cHL, UC, gastric/gastroesophageal junction cancer MSI-H tumors. Efficacy was not shown in multiple myeloma in combination with lenalidomide or pomalidomide and low-dose dexamethasone. Pembrolizumab as monotherapy or in combinations is being studied in various indications. For the treatment of unresectable or metastatic melanoma, pembrolizumab demonstrated superior efficacy over available treatment options (IPI, Investigator's choice chemotherapy) in participants with advanced melanoma who were treatment-naïve, as well as those who progressed on prior therapy, including ipilimumab. Pembrolizumab provided substantial, clinically meaningful benefits in OS, PFS, and ORR in participants with NSCLC who progressed after platinum-containing chemotherapy and whose tumor cells expressed PD-L1. In previously treated participants with PD-L1 TPS ≥1% and disease progression following platinum-containing chemotherapy, pembrolizumab provided a statistically significant and clinically meaningful OS benefit compared to standard docetaxel chemotherapy. For participants with previously untreated metastatic NSCLC whose tumors express high levels of PD-L1, pembrolizumab demonstrated significant improvements in PFS and OS over standard of care chemotherapy. Pembrolizumab in combination with pemetrexed/carboplatin for the first-line treatment of metastatic nonsquamous NSCLC demonstrated both a statistically significant and clinically meaningful difference in ORR and a statistically significant benefit in PFS compared with pemetrexed/carboplatin alone.  
For the treatment of advanced HNSCC in a heavily pretreated population, pembrolizumab demonstrated a clinically meaningful response rate and a prolonged duration of response that is substantially distinct from what is expected with standard of care in previously treated participants with HNSCC, and points to the meaningful clinical benefit of pembrolizumab. Pembrolizumab for the treatment of relapsed or refractory cHL, has demonstrated durable, robust, clinically meaningful responses in this heavily pretreated population that generally included standard front-line therapies, salvage therapies, auto-SCT if eligible with chemosensitive disease, other single agent or combination chemotherapy regimens as needed, and with or without brentuximab vedotin (BV). For the treatment of UC in participants who have not received prior chemotherapy and are cisplatin-ineligible, pembrolizumab demonstrated a clinically meaningful ORR in participants with locally advanced or metastatic UC. In participants with locally advanced or metastatic UC who have received platinum-containing chemotherapy, treatment with pembrolizumab demonstrated a significant improvement in OS and a clinically meaningful benefit in durable responses compared with standard of care therapies. In participants with MSI-H tumors, pembrolizumab provided evidence of clinically meaningful benefit over standard treatments, regardless of tumor histology. Data from early phase trials indicate that pembrolizumab has efficacy in mesothelioma with disease control rates of 72-77% and objective response rates of 20-21%. Likewise, the responses have been found to be durable, with the median duration of response being 12 months.  
[34, 35]
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1.2.6 rationale for combination therapy

The Pastan lab has constructed a murine breast cancer cell line expressing human mesothelin (66C14-M) which grows in BALB/c transgenic mice expressing human mesothelin. The immunotoxins SS1P (first generation anti-mesothelin immunotoxin) or RG7787 (also called LMB-100) were injected directly into established tumors and anti-CTLA-4 administered intraperitoneally. No cures occurred with CTLA-4 or immunotoxin SS1P/RG7787 alone. Combining anti-CTLA-4 with PBS, transient tumor shrinkage was observed in 1 of 6 mice and no complete remissions were obtained. However, combining anti–CTLA-4 and LMB-100 resulted in tumor size reduction in 11 of 13 mice, and complete tumor elimination in 8 of 13 mice (61%). Combining anti-CTLA-4 with SS1P resulted in the similar enhanced anti-tumor effects compared to single drug administration. LMB-100-I, an inactive mutant form of LMB-100. was tested as control. Only 1 of 13 mice achieved a complete remission when treated with LMB-100- I and anti–CTLA-4, suggesting cell killing by the immunotoxin is required for the antitumor effect (Figure 2). Mechanistic studies showed that combination treatment induced tumor regression was associated with increased numbers of tumor infiltrating CD8+ cells. Surviving mice were protected from tumor re-challenge by 66C14 cells not expressing mesothelin,  
 indicating the development of anti-tumor immunity.
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systemic lmb-100 administration plus anti-pd1

The Hassan lab has established a mouse syngeneic lung adenocarcinoma model expressing human mesothelin for studies of anti-mesothelin agents with immune-checkpoint inhibitors. Using this model, we observed increased anti-tumor efficacy of LMB-100 given systemically with anti-PD-1 antibody. As shown in Figure 3, LMB-100 was intravenously administered for 2 cycles (at a dose of 2.5mg/kg dose every other day for 3 times with cycle interval of 3 days). Anti-PD-1 antibody 10 mg/kg was administered i.p. twice with interval of 4 days between first and second dose. All the drug treatments were started when tumor size reached 100-120mm3.  
Combination therapy with LMB-100 and anti-PD-1 antibody resulted in greater tumor regression compared to treatment with LMB-100 or anti-PD-1 alone. Furthermore, treatment with LMB100 plus anti-PD-1 antibody significantly prolonged overall survival (74 days) compared to no drug treatment (38 days) or treatment with LMB-100 or anti-PD-1 antibody alone (52 days) (Figure 4). The precise mechanism of synergy is not understood at this time, but could be due to an abscopal effect from LMB-100.  
Version Date: *02/16/21* Figure 3: Combination therapy with LMB-100 plus anti-PD-1 antibody results in  
 synergistic anti-tumor efficacy in hMSLN expressing mouse syngeneic lung cancer model Figure 4: Treatment with LMB-100 plus anti-PD-1 antibody improves overall survival in  
 hMSLN expressing mouse syngeneic lung cancer model 1.2.7 Justification for Study Doses
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1.2.7.1 lmb-100

The planned LMB-100 dose is the recommended single agent dose from the phase 1 study of LMB-100 (NCT02798536) established at 140 mcg/kg, on days 1, 3 and 5 of a 21-day cycle. Only two cycles of LMB100 will be administered as it is anticipated that a majority of patients will develop neutralizing antibodies after 2 cycles of treatment.  
1.2.7.2 Pembrolizumab The planned dose of pembrolizumab for this study is 200 mg every 3 weeks (Q3W). Based on the totality of data generated in the Keytruda development program, 200 mg Q3W is the appropriate dose of pembrolizumab for adults across all indications and regardless of tumor type.  
As outlined below, this dose is justified by:
Clinical data from 8 randomized studies demonstrating flat dose- and exposure-efficacy relationships from 2 mg/kg Q3W to 10 mg/kg every 2 weeks (Q2W),  
Clinical data showing meaningful improvement in benefit-risk including overall survival at 200 mg Q3W across multiple indications, and Pharmacology data showing full target saturation in both systemic circulation (inferred from pharmacokinetic [PK] data) and tumor (inferred from physiologically-based PK
[PBPK] analysis) at 200 mg Q3W
Among the 8 randomized dose-comparison studies, a total of 2262 participants were enrolled with melanoma and non-small cell lung cancer (NSCLC), covering different disease settings (treatment naïve, previously treated, PD-L1 enriched, and all-comers) and different treatment settings (monotherapy and in combination with chemotherapy). Five studies compared 2 mg/kg Q3W versus 10 mg/kg Q2W (KN001 Cohort B2, KN001 Cohort D, KN002, KN010, and KN021), and 3 studies compared 10 mg/kg Q3W versus 10 mg/kg Q2W (KN001 Cohort B3, KN001 Cohort F2 and KN006). All of these studies demonstrated flat dose- and exposureresponse relationships across the doses studied representing an approximate 5- to 7.5-fold difference in exposure. The 2 mg/kg (or 200 mg fixed-dose) Q3W provided similar responses to the highest doses studied. Subsequently, flat dose-exposure-response relationships were also observed in other tumor types including head and neck cancer, bladder cancer, gastric cancer and classical Hodgkin Lymphoma, confirming 200 mg Q3W as the appropriate dose independent of the tumor type. These findings are consistent with the mechanism of action of pembrolizumab, which acts by interaction with immune cells, and not via direct binding to cancer cells. Additionally, pharmacology data clearly show target saturation at 200 mg Q3W. First, PK data in KN001 evaluating target-mediated drug disposition (TMDD) conclusively demonstrated saturation of PD-1 in systemic circulation at doses much lower than 200 mg Q3W. Second, a PBPK analysis was conducted to predict tumor PD-1 saturation over a wide range of tumor penetration and PD-1 expression. This evaluation concluded that pembrolizumab at 200 mg Q3W achieves full PD-1 saturation in both blood and tumor. Finally, population PK analysis of pembrolizumab, which characterized the influence of body weight and other participant covariates on exposure, has shown that the fixed-dosing provides similar control of PK variability as weight based dosing, with considerable overlap in the distribution of exposures from the 200 mg Q3W fixed dose and 2 mg/kg Q3W dose. Supported by these PK characteristics, and given that fixed-dose has advantages of reduced dosing complexity and reduced potential of dosing errors, the 200 mg Q3W fixed-dose was selected for evaluation across all pembrolizumab protocols.
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summary

In summary, our laboratory studies show remarkable synergy between LMB-100 and immune checkpoint inhibitors using local or systemic administration with anti-CTLA4 or anti-PD1 blocking antibodies. Based on clinical trials of pembrolizumab in subjects with treatment refractory pleural mesothelioma that show a response rate of 20% we propose a clinical trial of this combination to determine if administration of LMB-100 can improve the response rates seen with pembrolizumab alone i.e. improve response rate from 20% to 40%. Since the anti-tumor efficacy of pembrolizumab in patients with peritoneal mesothelioma is not know we have separated these patients into a separate cohort to get preliminary estimates of anti-tumor activity of this combination in this patient population.
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2 eligibility assessment and enrollment

2.1.1 Inclusion Criteria Participants are eligible to be included in the study only if all of the following criteria apply.  
2.1.1.1 Male and female participants who are at least 18 years of age on the day of signing the informed consent will be enrolled in the study.  
2.1.1.2 Subjects must have histologically confirmed diagnosis of:  
2.1.1.2.1 Cohort 1: Histologically confirmed epithelial or biphasic pleural mesothelioma (with
<50% sarcomatoid component) not amenable to potentially curative surgical resection. The diagnosis will be confirmed by the Laboratory of Pathology, CCR, NCI.  
OR
2.1.1.2.2 Cohort 2: Histologically confirmed epithelial or biphasic peritoneal mesothelioma
(with <50% sarcomatoid component) not amenable to potentially curative surgical resection. The diagnosis will be confirmed by the Laboratory of Pathology, CCR, NCI.  
2.1.1.3 Have provided archival tumor tissue sample or newly obtained core or excisional biopsy of a tumor lesion not previously irradiated. Formalin-fixed, paraffin embedded (FFPE) tissue blocks are preferred to slides. Newly obtained biopsies are preferred to archived tissue. Note: If submitting unstained cut slides, newly cut slides should be submitted to the testing laboratory within 14 days from the date slides are cut.
2.1.1.4 Have measurable disease based on RECIST 1.1 (see Section 6.3). Lesions in a previously irradiated area are considered measurable if progression has been demonstrated in such lesions.  
2.1.1.5 Subjects must have at least one prior chemotherapy regimen that includes pemetrexed and cisplatin or carboplatin.  
2.1.1.6 Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. See Appendix A. Evaluation of ECOG is to be performed within 7 days prior to start of study therapy.  
2.1.1.7 Have adequate organ and marrow function as defined below:  
| System                                                                                                                                                                                                                                                                                   | Laboratory Value                                                                                             |                                                                               |             |             |                |                |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------|-------------|-------------|----------------|----------------|
| Hematological - hemoglobin                                                                                                                                                                                                                                                               | ≥ 9 g/dL or ≥5.6 mmol/La                                                                                     |                                                                               |             |             |                |                |
| -                                                                                                                                                                                                                                                                                        | absolute neutrophil count                                                                                    | ≥ 1,500/mcL                                                                   |             |             |                |                |
| -                                                                                                                                                                                                                                                                                        | platelets                                                                                                    | ≥ 100,000/mcL                                                                 |             |             |                |                |
| Hepatic - total bilirubin                                                                                                                                                                                                                                                                | ≤ 2.5 X institutional ULN OR direct bilirubin ≤ ULN for participants with total bilirubin  levels >1.5 X ULN |                                                                               |             |             |                |                |
| -                                                                                                                                                                                                                                                                                        | AST and ALT                                                                                                  | ≤ 2.5 X institutional ULN (≤ 5 X ULN for  participants with liver metastases) |             |             |                |                |
| Renal - Creatinine OR - Measured or                                                                                                                                                                                                                                                      | calculatedb                                                                                                  |                                                                               |             |             |                |                |
| creatinine clearance (GFR  can also be used in place of  creatinine or CrCl)                                                                                                                                                                                                             | ≤1.5 × ULN OR ≥ 50 mL/min for participant with creatinine  levels > 1.5 X institutional ULN                  |                                                                               |             |             |                |                |
| Coagulation - International normalized ratio  (INR) OR prothrombin time  (PT) - Activated partial  thromboplastin time (aPTT)                                                                                                                                                            | ≤1.5 × ULN                                                                                                   | unless                                                                        | participant | is          | receiving      |                |
| anticoagulant therapy as long as PT or aPTT is  within therapeutic range of intended use of  anticoagulants                                                                                                                                                                              |                                                                                                              |                                                                               |             |             |                |                |
| ALT                                                                                                                                                                                                                                                                                      | (SGPT)=alanine                                                                                               | aminotransferase                                                              | (serum      | glutamic    | pyruvic        | transaminase); |
| AST (SGOT)=aspartate                                                                                                                                                                                                                                                                     | aminotransferase                                                                                             | (serum                                                                        | glutamic    | oxaloacetic | transaminase); |                |
| GFR=glomerular filtration rate; ULN=upper limit of normal. a. Criteria must be met without erythropoietin dependency and without packed red blood cell  (pRBC) transfusion within last 2 weeks.  b. Creatinine clearance (CrCl) or eGFR should be calculated per institutional standard. |                                                                                                              |                                                                               |             |             |                |                |
| 2.1.1.8                                                                                                                                                                                                                                                                                  | Must have left ventricular ejection fraction >50%.                                                           |                                                                               |             |             |                |                |  
2.1.1.8 Must have left ventricular ejection fraction >50%. 2.1.1.9 Must recovered from all AEs due to previous therapies to ≤Grade 1 or baseline.  
Participants with ≤Grade 2 neuropathy may be eligible. If participant received major surgery, they must have recovered adequately from the toxicity and/or complications from the intervention prior to starting study treatment.  
2.1.1.10 Must have recovered from all radiation-related toxicities, not require corticosteroids, and not have had radiation pneumonitis. A 1-week washout is permitted for palliative radiation (≤2 weeks of radiotherapy) to non-CNS disease.  
2.1.1.11 If participant received major surgery, they must have recovered adequately from the toxicity and/or complications from the intervention prior to starting study treatment.  
2.1.1.12 The effects of LMB-100 on the developing human fetus are unknown. For this reason and because anti-PD-1 antibodies such as pembrolizumab are assumed to be teratogenic:
2.1.1.12.1 A male participant must agree to use contraception as detailed in see Appendix B of this protocol during the treatment period and for at least 180 days after the last dose of study treatment and refrain from donating sperm 2.1.1.12.2 A female participant is eligible to participate if she is not pregnant (see Appendix B),
not breastfeeding, and at least one of the following conditions applies:
a. Not a woman of childbearing potential (WOCBP) as defined in Appendix B
OR
b. A WOCBP who agrees to follow the contraceptive guidance in Appendix B
during the treatment period and for at least 180 days after the last dose of study treatment.  
2.1.1.12.3 Should a woman become pregnant or suspect she is pregnant while she or her partner is participating in this study, she should inform her treating physician immediately.  
2.1.1.13 The participant provides written informed consent for the trial.  
2.1.2 Exclusion Criteria 2.1.2.1 Is currently participating in or has participated in a study of an investigational agent or has used an investigational device within 4 weeks prior to the first dose of study treatment.  
Note: Participants who have entered the follow-up phase of an investigational study may participate as long as it has been 4 weeks after the last dose of the previous investigational agent.  
2.1.2.2 Has known active CNS metastases and/or carcinomatous meningitis. Participants with previously treated brain metastases may participate provided they are radiologically stable, i.e. without evidence of progression for at least 4 weeks by repeat imaging (note that the repeat imaging should be performed during study screening), clinically stable and without requirement of steroid treatment for at least 14 days prior to first dose of study treatment.  
2.1.2.3 Has severe hypersensitivity (≥Grade 3) to pembrolizumab, LMB-100 and/or any of their excipients.  
2.1.2.4 Subjects who have received prior therapy with LMB-100, an anti-PD-1, anti-PD-L1, or anti-PD-L2 agent or with an agent directed to another stimulatory or co-inhibitory Tcell receptor (e.g., CTLA-4, OX-40, CD137).  
2.1.2.5 Has received prior systemic anti-cancer therapy including investigational agents within 4 weeks prior to start of study therapy.  
2.1.2.6 Has received prior radiotherapy within 2 weeks of start of study treatment. 2.1.2.7 Has had a prior pneumonectomy 2.1.2.8 Has received a live vaccine within 30 days prior to the first dose of study drug.  
Examples of live vaccines include, but are not limited to, the following: measles, mumps, rubella, varicella/zoster (chicken pox), yellow fever, rabies, Bacillus Calmette– Guérin (BCG), and typhoid vaccine. Seasonal influenza vaccines for injection are generally killed virus vaccines and are allowed; however, intranasal influenza vaccines
(e.g., FluMist®) are live attenuated vaccines and are not allowed.  
2.1.2.9 Has a diagnosis of immunodeficiency or is receiving chronic systemic steroid therapy
(in dosing exceeding 10 mg daily of prednisone equivalent) or any other form of immunosuppressive therapy within 7 days prior to start of study therapy.  
2.1.2.10 Has active autoimmune disease that has required systemic treatment in the past 2 years
(i.e. with use of disease modifying agents, corticosteroids or immunosuppressive drugs). Replacement therapy (e.g.., thyroxine, insulin, or physiologic corticosteroid replacement therapy for adrenal or pituitary insufficiency, etc.) is not considered a form of systemic treatment.  
2.1.2.11 Has a history of (non-infectious) pneumonitis/interstitial lung disease(ILD) that required steroids or has current pneumonitis/ILD
2.1.2.12 Has a history or current evidence of any condition, therapy, or laboratory abnormality that might confound the results of the study, interfere with the subject's participation for the full duration of the study, or is not in the best interest of the subject to participate, in the opinion of the treating investigator.  
2.1.2.13 Has known psychiatric or substance abuse disorders that would interfere with cooperation with the requirements of the trial.  
2.1.2.14 A WOCBP who has a positive urine pregnancy test within 72 hours prior to start of study therapy (see Appendix B). If the urine test is positive or cannot be confirmed as negative, a serum pregnancy test will be required. **Note:** in the event that 72 hours have elapsed between the screening pregnancy test and the first dose of study treatment, another pregnancy test (urine or serum) must be performed and must be negative in order for subject to start receiving study medication.  
2.1.2.15 Is pregnant or breastfeeding, or expecting to conceive or father children within the projected duration of the study, starting with the screening visit through 180 days after the last dose of trial treatment. Pregnant women are excluded from this study because LMB-100 + pembrolizumab are agents with the potential for teratogenic or abortifacient effects. Because there is an unknown but potential risk for adverse events in nursing infants secondary to treatment of the mother with LMB-100 + pembrolizumab, breastfeeding should be discontinued if the mother is treated with LMB-100 + pembrolizumab. These potential risks may also apply to other agents used in this study.  
2.1.2.16 Has a known history of Human Immunodeficiency Virus. 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. (Note: No HIV testing is required)
2.1.2.17 Has a known history of Hepatitis B (defined as Hepatitis B surface antigen [HBsAg]
reactive) or known active Hepatitis C virus (defined as HCV RNA [qualitative] is detected) infection. or active HBV or HCV infection. (Note: No testing for Hepatitis B and Hepatitis C is required.)
2.1.2.18 Has a known additional malignancy that is progressing or has required active treatment within the past 2 years. Note: Participants with basal cell carcinoma of the skin, squamous cell carcinoma of the skin, or carcinoma in situ (e.g. breast carcinoma, cervical cancer in situ) that have undergone potentially curative therapy are not excluded.  
2.1.2.19 Has an active infection requiring systemic therapy.  
2.1.3 Recruitment Strategies Information about the study will be posted on NIH sites such as clinicaltrials.gov and the CCR recruitment website. The study will also be publicized on various NIH social media platforms. Subjects will also be drawn from patients seen at the mesothelioma clinic at the NIH Clinical Center as well as from referrals from outside providers.
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2.2 screening evaluation

2.2.1 Screening activities performed prior to obtaining informed consent Minimal risk activities that may be performed before the subject has signed a consent include the following:  
Email, written, in person or telephone communications with prospective subjects Review of existing medical records to include H&P, laboratory studies, etc.  
Review of existing MRI, x-ray, or CT images Review of existing photographs or videos Review of existing pathology specimens/reports from a specimen obtained for diagnostic purposes A waiver of consent for these activities has been requested in section **12.6.1**.
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2.2.2 screening activities performed after

The following activities will be performed only after the subject has signed the study consent OR the consent for study 01-C-0129 (provided the procedure is permitted on that study) on which screening activities may also be performed. Assessments performed at outside facilities or on another NIH protocol within the timeframes below may also be used to determine eligibility once a patient has signed the consent. Performed at any time prior to start of study therapy 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 will be collected if archival tumor tissue is not available. Note: with the exception of collection of fresh biopsy, this activity is covered under the waiver referenced above and may be performed prior to obtaining consent.  
Performed within 28 days prior to start of study therapy or, if applicable, second course History and physical exam Vital signs including pulse oximetry ECG
Echocardiogram CT scan of chest, abdomen and/or pelvis and areas of known or suspected disease involvement; MRI may also be performed when appropriate. Additional imaging may be done as clinically indicated. (If patient being assessed for Second Course, please see Appendix D)
FDG-PET scan Urinalysis Performed within 10 days prior to start of study therapy or, if applicable, second course 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), lactate dehydrogenase, Thyroid (TSH, free T4, total T3)
Performed within 7 days prior to start of study therapy or, if applicable, second course ECOG performance status (within 7 days)
Performed within 72 hours prior to the start of study therapy or, if applicable, second course Urine or serum hCG in women of childbearing potential (must be repeated on C1D1 (pretreatment) if more than 72 hours have passed since screening assessment)
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2.3 participant registration and status

Registration and status updates (e.g. when a participant is taken off protocol therapy and when a participant is taken off-study) will take place per CCR SOP ADCR-2, CCR Participant Registration & Status Updates found here. 2.3.1 Treatment Assignment Procedures Cohorts  
| Number   | Name                    | Description                                                                                          |
|----------|-------------------------|------------------------------------------------------------------------------------------------------|
| 1        | Pleural mesothelioma    | Up to 12 or up to 25 (depending on interim analysis)  evaluable subjects with pleural mesothelioma   |
| 2        | Peritoneal mesothelioma | Up to 6 or up to 10 (depending on interim analysis) evaluable  subjects with peritoneal mesothelioma |
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arms

| Number   | Name                  | Description                                          |
|----------|-----------------------|------------------------------------------------------|
| 1        | LMB-100+pembrolizumab | LMB-100 administered in cycles 1 & 2 + pembrolizumab |
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stratifications randomization and arm assignment

No stratification or randomization will occur on the study. Subjects in Cohort 1 will be directly assigned to Arm 1. If 3 or more subjects among the first 12 enrolled have an objective response, 13 additional evaluable subjects will be enrolled. Subjects in Cohort 2 will be directly assigned to Arm 1. If 1 or more subjects among the first 6 enrolled have an objective response, 4 additional evaluable subjects will be enrolled.
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3 study implementation 3.1 study

This is an open-label, single center Phase II study of LMB-100 followed by pembrolizumab in patients with advanced pleural or peritoneal mesothelioma who have progressed on standard therapies. Patients will receive LMB-100 only at the single agent MTD, 140 mcg/kg*, on days 1, 3 and 5 of a 21-day cycle for the first two cycles. Regardless of tumor response (CR/PR, SD, PD) after LMB-100 monotherapy, if conditions in section 3.3.2.1 are met, subjects will receive pembrolizumab monotherapy (200 mg) on day 1 of each subsequent 21-day cycle for up to 2 years or until disease progression on pembrolizumab, or intolerable toxicity, whichever occurs first. Cycle 3 dosing will not be initiated until LMB-100 related toxicities have resolved to grade 1 or better; however, if the initiation of cycle 3 is delayed for more than 4 weeks, the patient will be removed from study therapy. If needed a dose level below the single agent MTD of LMB-100 will be explored. Tumor biopsies will be performed at baseline, at the end of cycle 2 and at the end of cycle 4 to evaluate changes in the tumor immune microenvironment following treatment with LMB-100 and pembrolizumab. * Note: A weight-based dose cap will be applied. LMB-100 dose for patients weighing more than 100 kg will be calculated as if they weigh 100kg.
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3.1.1 schema

3-week cycles; Response evaluation every 2 cycles; biopsy at baseline, at the end of cycle 2 and at the end of cycle 4. Pembrolizumab may continue for a maximum of 2 years. All participants who stop study treatment with stable disease or better may be eligible for an additional 17 cycles of pembrolizumab if they progress after stopping study treatment from the initial phase.
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3.1.2 second course

All participants who stop study treatment with stable disease or better may be eligible for up to an additional 17 cycles (approximately 1 year) of pembrolizumab treatment if they progress after stopping study treatment from the initial treatment phase. This retreatment is termed the Second Course Phase of this study and is only available if the study remains open and the participant meets the following conditions:
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either

Stopped initial treatment with study treatment after attaining an investigator-determined confirmed CR based on RECIST 1.1, and o Was treated with at least 8 cycles of study treatment before discontinuing treatment, and  
o Received at least 2 treatments with pembrolizumab beyond the date when the initial CR was declared
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or

Had SD, PR, or CR and stopped study treatment after completion of 35 administrations
(approximately 2 years) of study treatment for reasons other than disease progression or intolerability
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and

Experienced an investigator-determined radiographic disease progression by RECIST 1.1 after stopping initial treatment, and  
o No new anticancer treatment was administered after the last dose of study treatment, and
o The participant meets all of the safety parameters listed in the inclusion criteria and none of the safety parameters listed in the exclusion criteria, and
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o the study is ongoing

An objective response or disease progression that occurs during the Second Course Phase for a participant will not be counted as an event for the safety and efficacy endpoints of the study.
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3.2 drug administration 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 (see section 3.1 for dose) will be given as an IV solution on Days 1, 3, and 5 (QOD× 3) of a 21-day cycle for two cycles. 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.
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3.2.1.1 preparation instructions

1. Visually inspect the LMB-100 drug product prior to dose preparation. The solution should be colorless to brownish and may contain a small amount of translucent-to white amorphous LMB-100 particulates. Do not use if other particulate matter is seen or if the study drug is discolored. Do not shake the vial to mix.  
2. Use an appropriate sized syringe and needle to draw up the required volume of LMB-100
(undiluted drug) plus an additional 3 mL (overfill for priming the syringe extension line) from the vial.  
3. Attach a Smiths Medical Ultra™ Small Bore Extension Set (REF \#MX448HL60) to the syringe and a Smith medical 0.2 micron filter set (MX448HF) to the end of the extension set, then prime close to the distal end. Purge all air from the syringe and line. Cap with a sterile Spiro cap before dispensing.  
3.2.1.2 Administration Instructions 1. Intravenous (IV). Do not administer as IV push or bolus. 2. LMB-100 is administered either peripherally or centrally through a patient's vascular access device. If there is no pre-existing central vascular access device (VAD) and peripheral access is inadequate, a central access device will be installed.  
3. Undiluted LMB-100 (1 mg/mL) will be transferred to a disposable syringe and administered by IV infusion using a syringe pump. LMB-100 will be diluted in-line 1:10 with 0.9% Sodium Chloride Injection, USP immediately prior to administration.  
4. To accomplish this, a side flow with 0.9% Sodium Chloride Injection, USP, must be applied. A 4-way stopcock (alternatively two 3-way stopcocks joined end-to-end, or an alternative manifold with at least two inlets and one outlet ports that can be opened simultaneously) should be positioned at the patient's VAD to allow simultaneous
infusion of LMB-100 and 0.9% Sodium Chloride Injection, USP. The infusion rate of the 0.9% Sodium Chloride Injection will be 9-times the hourly rate of LMB-100.  
  
The LMB-100 drug product should be filtered using an in-line filter as depicted.  
If using a peripheral IV, the IV line should remain in place for 2 hours after the end of the infusion. 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. 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 DP. The infusion duration should be 30 minutes (-5 minutes, + 10 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 3.3.1.
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3.2.1.3 premedications for patients receiving

Due to the prevalence of infusion related reactions (IRRs) seen in the previous study of LMB100, all patients will be predicated 30-60 minutes (+ 30 minutes) prior to each LMB-100 administration with the following medications:  
| Dose (mg)                                       | Route   |              |
|-------------------------------------------------|---------|--------------|
| Acetaminophen                                   | 650     | Orally       |
| Ranitidinea                                     | 150     | Orally       |
| Diphenhydramineb                                | 25-50   | Orally or IV |
| a or alternative H2 blocker at an adequate dose |         |              |  
a or alternative H2 blocker at an adequate dose b or alternative antihistamine at an adequate dose.  
(See section 3.3.1 for complete instructions on response to IRRs)
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3.2.2 pembrolizumab

Pembrolizumab 200 mg will be administered as a 30-minute IV infusion every 3 weeks = 1 cycle. Pembrolizumab will be administered on day 1 (± 3 days) of each cycle starting with cycle 3 (if conditions in section 3.3.2.1 are met) for up to 2 years unless criteria for removal from therapy (3.5.1) are met. Note: As indicated in section 3.1.2, in certain circumstances the total duration of pembrolizumab treatment may exceed the 2-year limit. Every effort will be made to target infusion timing to be as close to 30 minutes as possible. However, given the variability of infusion pumps, a window of -5 minutes and +10 minutes is permitted (i.e., infusion time is 30 minutes: -5 min/+10 min). Pembrolizumab may be administered on an outpatient basis. The Pharmacy Manual contains specific instructions for the preparation of the pembrolizumab infusion fluid and administration of infusion solution.
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3.3 dose modifications/delays 3.3.1 lmb-100

LMB-100 infusion may be held for up to 72 hours due to drug-associated toxicity or adverse events from other intercurrent medical conditions (such as primary cancer diagnosis) that resolve with or without medical intervention to grade 2 or less within this time frame. Toxicities for which further LMB-100 treatment should NOT be given or which are exceptions to the above guidelines are stated below in Table 4. The table also provides guideline on how to manage some toxicities anticipated with LMB-100.  
| Table 4. 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 o Patients experiencing IRR CTCAE Grade 4 or  anaphylaxis must be permanently discontinued from  LMB-100 treatment   |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| Version Date: 02/16/21 Event   | Action to Be Taken                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |    |    |                                                  |
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| Capillary leak syndrome        | In the event of Grade ≥2 CTCAE capillary 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 the  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 |    |    |                                                  |
| Inflammatory                   | reactions                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  | to | o  | Hydrocortisone (200 mg IV) or equivalent dose of |
| serosal membranes              | another 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  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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |    |    |                                                  |  
| Event                                                                                                      | Action to Be Taken 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                                                                                                                                                                         |
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| Renal Toxicity                                                                                             | In the event of Grade 1 or greater renal toxicity consider increasing  oral or intravenous hydration, and consider delaying LMB-100  administration by up to 72 hours. In the event of Grade 2 or greater renal toxicity hold LMB-100  administration until recovery to Grade 1 or better. If this does not  occur within 72 hours, no further LMB-100 should be given during  the cycle. |
| IRR = infusion related reaction; IV = intravenous; CTCAE = Common Terminology Criteria  for Adverse Events |                                                                                                                                                                                                                                                                                                                                                                                           |
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3.3.2 pembrolizumab

3.3.2.1 Dose delays Cycle 3 day 1 of pembrolizumab may be delayed for up to 4 weeks if LMB-100 related toxicity has not resolved to grade 1 or better. If the delay exceeds 4 weeks, the patient will be removed from study therapy.  
3.3.2.2 Dose Modification and toxicity management for immune-related AEs associated with pembrolizumab AEs associated with pembrolizumab exposure may represent an immunologic etiology. These immune-related AEs (irAEs) may occur shortly after the first dose or several months after the last dose of pembrolizumab treatment and may affect more than on body system simultaneously. Therefore, early recognition and initiation of treatment is critical to reduce complications. Based on existing clinical study data, most irAEs were reversible and could be managed with interruptions of pembrolizumab, administration of corticosteroids and/or other supportive care. For suspected irAEs, ensure adequate evaluation to confirm etiology or exclude other causes. Additional procedures or tests such as bronchoscopy, endoscopy, skin biopsy may be included as part of the evaluation. Based on the severity of irAEs, withhold or permanently discontinue pembrolizumab and administer corticosteroids. Dose modification and toxicity management guidelines for irAEs associated with pembrolizumab are provided in Table 5. Table 5: Dose modification and toxicity management guidelines for immune related AEs