untitled h1
untitled h2

Abbreviated Title: LMB-100 plus pembro in NSCLC NIH Protocol \#: 19C0127 Version Date: December 3, 2021 NCT Number: NCT04027946 Title: A Phase II Study of LMB-100 Followed by Pembrolizumab in the Treatment of Adults with Mesothelin-Expressing Non-Squamous Non-Small Cell Lung Cancer (NSCLC)  
NCI Principal Investigator: Azam Ghafoor, MD  
Thoracic & GI Malignancies Branch (TGMB) Center for Cancer Research (CCR), NCI, NIH Building 10, Room 4-5330 9000 Rockville Pike Bethesda, MD 20892 Phone: 240-858-3289 E-mail: azam.ghafoor@nih.gov  
| Drug Name:    | LMB-100             | Pembrolizumab          | Mesothelin     |                |
|---------------|---------------------|------------------------|----------------|----------------|
| (Keytruda®)   | Expression  Testing | TruSight  Oncology 500 |                |                |
| IND Number:   | 123332              | 123332                 | NSR device     | NSR device     |
| Sponsor:      | CCR, NCI, NIH       | CCR, NCI, NIH          | CCR, NCI, NIH  | CCR, NCI, NIH  |
| Manufacturer: | Selecta             | Merck                  | NCI Laboratory | NCI Laboratory |
| Biosciences   | of Pathology        | of Pathology           |                |                |
| Supplier:     | CC Pharmacy         | NIH CC                 | NCI Laboratory | NCI Laboratory |
| Pharmacy      | of Pathology        | of Pathology           |                |                |
untitled h1
precis background

Mesothelin is expressed in approximately half of all lung adenocarcinomas.  
LMB-100 has demonstrated anti-tumor efficacy against several mesothelin expressing tumor models including non-small cell lung cancer (NSCLC).  
Programmed death ligand 1 (PD-1) 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.  
Pembrolizumab, an IgG4 monoclonal antagonist antibody to PD-1, is FDA approved in the frontline for advanced non-squamous NSCLC as a single agent with high PD-L1 expression [tumor proportion score (TPS) ≥50%] or in combination with platinum-based doublet chemotherapy (PD-L1 unselected). It also approved in the second-line for high PDL1 expressing tumors (TPS ≥ 1%).  
Combination treatment with LMB-100 plus pembrolizumab results in greater anti-tumor efficacy in murine lung cancer model.
Objectives:  
To determine the objective response rate of LMB-100 followed by pembrolizumab in the treatment of subjects with mesothelin-expressing non-squamous non-small cell lung cancer (NSCLC) previously treated with immune checkpoint inhibitors.
Eligibility:  
Histologically confirmed locally advanced or metastatic non-squamous, non-small cell lung cancer lacking an EGFR sensitizing mutation, ALK or ROS1 gene rearrangement and not amenable to potentially curative surgical resection or chemoradiation.  
Tumor mesothelin expression of at least 25% of tumor cells as determined by the Laboratory of Pathology at the NCI.  
Subjects must have at least progressed after one prior platinum-based doublet chemotherapy AND standard immune checkpoint inhibitor (ICI) with either frontline single-agent pembrolizumab, or in combination with platinum-based doublet chemotherapy, or second-line single-agent nivolumab, pembrolizumab, or atezolizumab.  
Age ≥ 18 years.
untitled h1
design

This is an open-label, single center phase II study of LMB-100 followed by pembrolizumab in subjects with mesothelin expressing NSCLC who have progressed on standard therapies Subjects will receive LMB-100 at the single agent MTD (140mg/kg) on days 1, 3 and 5 of a 21-day cycle for up to 2 cycles and pembrolizumab 200 mg on day 1 of cycle 3 of a 21day cycle (or cycle 2 if disease progression is observed after 1 cycle) onwards until disease progression (on or after pembrolizumab) or intolerable toxicity for a maximum of 2 years (unless second course initiated).
untitled h1
design

The total accrual ceiling for the screening will be set at 100 total patients in order to treat 23 subjects.  
3  
| TABLE OF CONTENTS                                                                                                                                  |                                                                                                                                                      |
|----------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------|
| PRÉCIS                                                                                                                                             |                                                                                                                                                      |
| TABLE OF CONTENTS ...............................................................................................................................4 |                                                                                                                                                      |
| STATEMENT OF COMPLIANCE                                                                                                                            |                                                                                                                                                      |
| l                                                                                                                                                  | INTRODUCTION                                                                                                                                         |
| 1.1                                                                                                                                                | Study Objectives .                                                                                                                                   |
| 1.2                                                                                                                                                | Background and Rationale ..........................................................................................................8                 |
| 2                                                                                                                                                  | ELIGIBILITY ASSESSMENT AND ENROLLMENT ...................................................................28                                          |
| 2.1                                                                                                                                                | Eligibility Criteria .............................................................................................................................28 |
| 2.2                                                                                                                                                | Screening Evaluation                                                                                                                                 |
| 2.3                                                                                                                                                | Participant Registration and Status Update Procedures ...................................................35                                          |
| 2.4                                                                                                                                                | Treatment Assignment Procedures                                                                                                                      |
| 3                                                                                                                                                  | STUDY IMPLEMENTATION                                                                                                                                 |
| 3.1                                                                                                                                                | Study Design ...........................................................................................................................35           |
| 3.2                                                                                                                                                | Drug Administration ................................................................................................................37               |
| 3.3                                                                                                                                                | Dose Modifications/Delays                                                                                                                            |
| Study Calendar .....................................................................................................................49             |                                                                                                                                                      |
| 3.4                                                                                                                                                |                                                                                                                                                      |
| 3.5                                                                                                                                                | Cost and Compensation                                                                                                                                |
| Criteria for Removal from Protocol Therapy and Off Study Criteria ..........................53                                                     |                                                                                                                                                      |
| 3.6                                                                                                                                                |                                                                                                                                                      |
| 4                                                                                                                                                  | CONCOMITANT MEDICATIONS/MEASURES ...........................................................................54                                       |
| s                                                                                                                                                  | Correlative Studies for Research                                                                                                                     |
| Biospecimen Collection .                                                                                                                           |                                                                                                                                                      |
| 5.1                                                                                                                                                |                                                                                                                                                      |
| 5.2                                                                                                                                                | Sample Collection schedule ...........................................................................................................62             |
| 5.3                                                                                                                                                | Sample Storage, Tracking and Disposition .........................................................................63                                 |
| 5.4                                                                                                                                                | Samples for Genetic/Genomic Analysis .................................................................................65                             |
| 6                                                                                                                                                  | DATA COLLECTION AND EVALUATION ..................................................................................66                                  |
| DATA COLLECTION                                                                                                                                    |                                                                                                                                                      |
| 6.1                                                                                                                                                |                                                                                                                                                      |
| 6.2                                                                                                                                                | DATA Sharing Plans ...............................................................................................................................67 |
| 6.3                                                                                                                                                | Response Criteria ............................................................................................................................67     |
| TOXICITY Criteria                                                                                                                                  |                                                                                                                                                      |
| 6.4                                                                                                                                                |                                                                                                                                                      |
| 7                                                                                                                                                  | NIH REPORTING REQUIREMENTS/DATA SAFETY MONITORING PLAN...............76                                                                              |
| 7.1                                                                                                                                                | Definitions                                                                                                                                          |  
| 7.2   | OHSRP Office of Compliance and Training / IRB Reporting 77            |
|-------|-----------------------------------------------------------------------|
| 7.3   | NIH Required Data and Safety Monitoring Plan 77                       |
| 8     | SPONSOR SAFETY REPORTING 78                                           |
| 8.1   | Definitions 78                                                        |
| 8.2   | Assessment of Safety Events 79                                        |
| 8.3   | Reporting of Serious Adverse Events 79                                |
| 8.4   | Waiver of expedited reporting to CCR 80                               |
| 8.5   | Reporting Pregnancy 80                                                |
| 8.6   | Regulatory Reporting for Studies Conducted Under CCR-Sponsored IND 80 |
| 9     | CLINICAL MONITORING 81                                                |
| 10    | STATISTICAL CONSIDERATIONS 81                                         |
| 10.1  | Statistical Hypothesis 81                                             |
| 10.2  | Populations for Analyses 82                                           |
| 10.3  | Statistical Analyses 82                                               |
| 11    | HUMAN SUBJECTS PROTECTIONS 84                                         |
| 11.1  | Rationale For Subject Selection 84                                    |
| 11.2  | Participation of Children 84                                          |
| 11.3  | Participation of Subjects Unable to Give Consent 84                   |
| 11.4  | Evaluation of Benefits and Risks/Discomforts 85                       |
| 11.5  | Risks/Benefits Analysis 86                                            |
| 11.6  | Consent Process and Documentation 86                                  |
| 12    | REGULATORY AND OPERATIONAL CONSIDERATIONS 87                          |
| 12.1  | Study Discontinuation and Closure 87                                  |
| 12.2  | Quality Assurance and Quality Control 87                              |
| 12.3  | Conflict of Interest Policy 88                                        |
| 12.4  | Confidentiality and Privacy 88                                        |
| 13    | PHARMACEUTICAL AND INVESTIGATIONAL DEVICE INFORMATION 89              |
| 13.1  | Mesothelin testing (NSR device) 89                                    |
| 13.2  | TruSight Oncology 500 Assay (NSR device) 89                           |
| 13.3  | LMB-100 (IND #123332) 90                                              |
| 13.4  | Pembrolizumab (Keytruda®) 96                                          |
| 14    | REFERENCES 104                                                        |  
| 15                                                                                    | Appendices 108                                                                   |
|---------------------------------------------------------------------------------------|----------------------------------------------------------------------------------|
| 15.1                                                                                  | Appendix A: Performance Status Criteria 108                                      |
| 15.2                                                                                  | Appendix B: Contraception Requirements 109                                       |
| 15.3                                                                                  | Appendix C: Imaging and Treatment after First Radiologic Evidence of Progressive |
| Disease on Pembrolizumab 111 15.4 Appendix D: Second Course (Retreatment) Imaging 112 |                                                                                  |
untitled h1
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.
untitled h1
1 introduction 1.1 study objectives

1.1.1 Primary Objective Determine the objective response rate of LMB-100 followed by pembrolizumab in the treatment of subjects with mesothelin-expressing non-squamous non-small cell lung cancer (NSCLC) previously treated with immune checkpoint inhibitors.  
1.1.2 Secondary Objectives Determine duration of overall response, progression free survival and overall survival. Determine the safety and tolerability of LMB-100 followed by pembrolizumab in the treatment of subjects with mesothelin-expressing non-squamous non-small cell lung cancer (NSCLC) previously treated with immune checkpoint inhibitors.  
1.1.3 Exploratory Objectives Establish the correlation of response with tumor mesothelin expression.  
Evaluate correlation of tumor response with tumor PD-L1 expression. Evaluate changes in the tumor microenvironment following treatment with LMB-100 followed by pembrolizumab.  
Evaluate the utility of serum mesothelin and megakaryocyte potentiating factor (MPF) as a biomarkers of tumor response.  
Define the pharmacokinetics characteristics of LMB-100 to correlate responses with LMB100 blood levels.  
Determine the incidence of antibody development at the end of cycles 1 and 2 with this combination.  
Correlation of incidence of KRAS mutation with mesothelin immunohistochemical expression in tumor samples.  
Determine the utility of circulating tumor DNA (ctDNA) and characteristic mutations for biomarkers analysis of response in blood, and overall tumor genome evolution over the course of treatment.  
Establish cell lines and cytology studies
To assess changes in circulating immune cell and cytokine levels caused by the treatment  
1.2 BACKGROUND AND R**ATIONALE**
untitled h1
1.2.1 study disease

234,000 new lung cancer cases are diagnosed every year in the US alone and the leading cause of cancer mortality worldwide. Approximately 80% of lung cancers are classified as NSCLC; a collection of histologically heterogeneous neoplasms including adenocarcinomas and squamous cell carcinoma subtypes(1). Patients with stage I-III NSCLC are generally treated with curative surgery, chemotherapy, radiation therapy, or a combined modality. Although patients with an early stage disease will benefit from these modalities, most patients (~40-45%) will present with more advanced stage IV disease at diagnosis or will progress after failing definitive treatment. For patients with advanced disease, the 5- year survival rate is less than 10% (2). Current standard systemic treatment involves an integration of platinum-based chemotherapy, targeted agents, and immunotherapy depending on histology (squamous versus non-squamous) and presence or absence of EGFR sensitizing mutations, ALK or ROS1 gene rearrangements, and expression of programmed death ligand 1
(PD-L1) by immunohistochemistry (IHC). Frontline treatment for advanced or stage IV nonsquamous NSCLC without an EGFR-sensitizing mutation of ALK or ROS1 gene rearrangement and performance status (PS) 0 or 1(3):  
High PD-L1 expression [tumor proportion score (TPS) >= 50%] single agent pembrolizumab Low PD-L1 expression (TPS<50%) pemetrexed and platinum chemotherapy with pembrolizumab
Second-line treatment (initial cytotoxic therapy) for advanced tumors lacking sensitizing EGFR mutation or ALK or ROS1 translocation and PS 0 or 1(3):  
High PD-L1 expression (TPS >=1%) and have not received prior immune therapy single agent nivolumab, pembrolizumab, or atezolizumab Low or unknown PD-L1 (TPS <1%) and have not received prior immune therapy: nivolumab or atezolizumab Systemic therapy with docetaxel or pemetrexed Platinum combination chemotherapy with pemetrexed if received immune checkpoint inhibitor initially.
1.2.2 Mesothelin as a target for Lung Adenocarcinoma Mesothelin is a 40kDa 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.(4) 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 lung adenocarcinomas.(5) 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.(6) Ho et al, initially reported that in a cohort of lung adenocarcinomas (n=12), 10/12 patients or 86% showed expression of mesothelin mRNA. (7) In addition, 82% of the patients showed expression of the mesothelin precursor protein and half with mature form. Lung adenocarcinomas showed strong and diffuse staining for mesothelin versus weak-modest staining in squamous. Thomas et al further observed MSLN expression in 53% of advanced (stages IIIB to IV) lung adenocarcinomas, with high expression (>25% of mesothelin positive cells) found in approximately 24% of patients.(8) Importantly, high mesothelin expression was associated with inferior survival (median 18.2 months vs. 32.9 months; P = 0.014). Another study from Memorial Sloan Kettering Cancer Center demonstrated that mesothelin was expressed around 70% by IHC in stage I-III lung adenocarcinomas. (8) Interestingly, increased expression was correlated with reduced overall survival (OS) through a multivariate analysis (HR 1.78, p<0.01). In unpublished data collected at the NCI by Thomas et al, it was demonstrated that 10% of patients with lung adenocarcinoma co-express PD-L1 and mesothelin expression (>25% of cells). (**Table 1**; Unpublished NCI data). 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.(9) The Laboratory of Molecular Biology (LMB) and the Thoracic and GI Malignancies Branch (TGMB), CCR, NCI have pioneered the use of mesothelin-targeted agents and clinical trials over the last decade. Given that mesothelin is expressed in half of lung adenocarcinomas (co-expressed in 28% of cells with PD-L1) and is associated with a worse prognosis, there is an unmet need to utilize mesothelin directed therapies in the setting of mesothelin expressing NSCLC which may enhance overall responses to immunotherapy.  
Table 1: Co-expression of PD-L1 and Mesothelin. Patients with advanced (stage III/IV) lung adenocarcinoma  
  
enrolled in a prospective trial of molecular profiling at the National Cancer Institute with IHC performed for mesothelin and PD-L1 (n= 71) (unpublished NCI data).
untitled h1
1.2.3 lmb-100

Recombinant immunotoxins (RIT) 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.(10) It has anti-tumor efficacy against several mesothelin expressing tumor models including mesothelioma PDX models.(11) Also, the debut of mesothelin-targeting chimeric antigen receptor (CAR-T) cells has led to Phase I/II clinical trials of targeting mesothelin expressing lung cancers by administering autologous-engineered T cells systemically or intra-pleurally (NCT02414269). A phase I clinical trial of LMB-100 in patients with mesothelioma with or without nab-paclitaxel chemotherapy has been completed at NCI where the maximum tolerated dose (MTD) has been established and will be used in combination studies with pembrolizumab. Single agent activity was also evaluated, however, there is no single agent activity of LMB-100 in mesothelin expressing non-small cell lung cancers. LMB-100 is also currently being evaluated in other mesothelin expressing cancers such as pancreatic cancer.
untitled h1
1.2.3.1 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 kDa 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.(12, 13) 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.  
Figure 1. Structural models of SS1P and its de-immunized variants SS1P-LR and LMB-100. The targeting domain consists of VL (cyan) and VH (magenta). The linker between the targeting domain and PE contains the furin cleavage site (green), which is required for toxin cytotoxic activity. The furin cleavage site is part of PE Domain II. The remainder of Domain II (gray) is unnecessary for cytotoxicity and has been deleted in the PE24-based toxins, SS1-LR and LMB-100. Domain III (yellow) is the catalytic domain of PE. In LMB-100, alanine point mutations were introduced at seven bulky hydrophilic residues (red) to silence human B cell epitopes within this domain. Deletion of Domain II reduces the size of the molecule into the range where it can be easily filtered by the kidneys, reducing serum half-life. LMB-100 contains a larger humanized Fab for targeting which raises its molecular weight above this threshold.
untitled h1
1.2.3.2.1 nonclinical pharmacology

In vitro LMB-100 inhibited viability of a variety of mesothelin-positive cancer cell lines, including NSCLC cell line, 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 mesothelinexpressing cancer cell lines and patient derived xenograft models.(10, 14)
Animal studies demonstrated that a single cycle of LMB-100 treatment given at an optimal dose of approximately 2 mg/kg, 3  per week, every other day (QOD) achieved tumor regressions in subcutaneous xenografts of adenosquamous lung carcinoma (H596) in severe combined immunodeficient (SCID) beige mice. Three consecutive treatment cycles, given with 1 week breaks in between, led to massive shrinkage of large tumors with an average initial volume of 600 mm3. Tumor regressions in monotherapy were also achieved when treating subcutaneous xenografts of mesothelioma (NCI-H226), gastric (MKN-28), and triple negative breast (HCC70) cancer cell lines in athymic nude mice. Highly synergistic antitumor efficacy was observed in combination therapy with paclitaxel when treating subcutaneous xenografts of the recombinant high mesothelin expressing A431/H9 cell line or the pancreatic cancer cell line KLM1. Synergy was also observed in the HCC70 and MKN-28 cell lines. These results support evidence that LMB100 in monotherapy or in combination with standard chemotherapies may provide clinical benefit to patients with cancer.  
1.2.3.2.2 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. Toxicokinetics after repeated IV dosing in cynomolgus monkeys demonstrated an increase in total exposure in a dose proportional manner between 0.1 mg/kg and 3.0 mg/kg. No accumulation was observed over 5 consecutive days of treatment or over two dosing cycles with 3  per week dosing. Almost all monkeys developed ADAs upon treatment, while induction of high ADA levels impaired the exposure of free drug. In some cases, the induction of ADAs may have induced a slight increase in exposure The relationship between systemic drug exposure and anti-tumor activity of LMB-100 was investigated on human lung cancer NCI-H596 xenograft growth in female SCID beige mice. Free and total drug profiles were similar in mice. Modeling estimated a plasma concentration of 6800 ng/mL ( 36%) to trigger a half maximal rate of tumor regression. Concentrations of LMB-100 above this level resulted in potent tumor regression after dosing. Normalized for exposure, SS1P was found to be ~3-fold more potent than LMB-100 in terms of tumor growth inhibition.
untitled h1
1.2.3.2.3 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 mcg/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 mcg/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.
untitled h1
1.2.3.3 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 MSLNexpressing metastatic or locally advanced solid tumors for whom no standard therapy was available. Secondary objectives included determination of the RP2D and schedule, exploration of preliminary anti-tumor activity by assessing objective response rate (ORR) and disease control rate (DCR), and assessment of pharmacodynamic effects. A total of 15 participants were enrolled onto the study before termination. Median age of participants was 60.8 years and 53.3% were female. All participants had received prior anti-cancer therapy for their tumors. Enrolled participants had advanced mesothelioma (7), ovarian cancer (3), pancreatic cancer (3), and gastroesophageal cancer (2). Tumors from 13 of the 15 participants treated had moderately to strongly positive MSLN expression as measured by central IHC analysis.  
LMB-100 was administered intravenously on Days 1, 3 and 5 of a 21-day treatment cycle. No premedications were given. Treatment was initiated at the MTD of SS1P, 45 mcg/ kg. Five different dose levels were tested (see **Table 2**). 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.  
  
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. 1.2.3.4 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 mesotheliomas; 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. 1.2.3.5 Infusion-Related Reactions and Hypersensitivity Including Anaphylaxis LMB-100 administration may cause infusion-associated symptoms such as fever, chills, hypotension, shortness of breath, skin rash, headache, nausea, and/or vomiting. Such reactions typically occur during or shortly after an infusion, predominantly the first infusion. Patients may also develop IgE-mediated hypersensitivity reactions to LMB-100. IRRs may be indistinguishable from an anaphylactic reaction. Patients should receive full supportive care to treat IRRs or anaphylaxis according to institutional practice. If infusion-associated signs or symptoms occur, patients should be monitored until complete resolution. In vitro data suggest that the risk for the release of pro-inflammatory cytokines upon first administration of LMB-100 to humans is low (human whole blood assay, see section **1.2.3.2.3**). Past experience with monoclonal antibodies that demonstrated a risk in the whole blood assay has shown that this risk could be effectively managed in the clinic with appropriate risk-minimization measures. The release of pro-inflammatory cytokines is believed to be partially responsible for the occurrence of IRRs. Free LMB-100 plasma concentrations were measured with a validated ELISA with a lower limit of quantification of 2.1 ng/mL. Doses ranged from 140 mcg/kg - 170 mcg/kg. Samples for pharmacokinetic (PK) analysis were obtained from patients at pre-dose, end of infusion (EOI; 30min post start), and 1 hour, 2 hours, 3 hours, 4 hours and 6 hours post EOI. Concentration data for each dose was plotted over time to assess the impact of increasing anti-drug antibodies (ADAs) that are generated in response to LMB-100 exposure. 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. 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.  
1.2.3.5.2 Anti-Drug Antibodies (ADAs) and LMB-100 Drug Levels Twelve participants were evaluable for efficacy. The best confirmed overall response was stable disease in 3 participants. A Roche-developed ELISA test was used to retrospectively assess antidrug antibody (ADA) titers. 5 of 15 participants had detectable ADAs at study enrollment while the remaining participants did not, however, the remaining participants developed detectable ADAs by the end of Cycle 2. Immunogenicity of LMB-100 did affect serum drug levels. All evaluable participants achieved expected serum drug levels during the first cycle of treatment. Six of 7 participants without pre-existing ADAs achieved effective drug levels during the second cycle, while 0 of 2 participants with pre-existing ADAs did. One of 3 participants that received a third cycle of treatment also achieved effective drug levels during this cycle. A positive test for ADAs did not definitively predict poor blood levels in the subsequent cycle (see patient 1101 in Table 3). In summary these data show that the presence of ADAs is not predictive of ability to achieve measurable LMB-100 concentration in the serum, which is the most important parameter for drug efficacy.  
Cycle 1 Cycle 2 Cycle 3  
Patient Dose  
(mcg/kg)  
ADA (Day1)  
Cmax  
(ng/ml)  
ADA (Day1)  
Cmax (ng/ml)  
dC1/dC (%)  
ADA (Day1)  
Cmax  
(ng/ml)  
dC1/dC (%)  
1002 65 0 1150 8100 711 -38 - - - 1401* 100 0 1790 0 1610 -10 24300 267 -85 1402 100 0 1650 0 1360 -18 8100 0 -100 1101* 170 0 2760 300 3950 43 900 3490 26 1202 170 0 3040 0 1940 -36 - - - 1301 170 0 3430 72900 527 -85 - - - 1403 170 0 1930 900 1550 -20 - - -  
| Cycle 1                                                                                                                                    | Cycle 2   | Cycle 3   |        |         |      |        |         |      |        |
|--------------------------------------------------------------------------------------------------------------------------------------------|-----------|-----------|--------|---------|------|--------|---------|------|--------|
| Patient                                                                                                                                    | Dose      | ADA       | Cmax   | ADA     | Cmax | dC1/dC | ADA     | Cmax | dC1/dC |
| (mcg/kg)                                                                                                                                   | (Day1)    | (ng/ml)   | (Day1) | (ng/ml) | (%)  | (Day1) | (ng/ml) | (%)  |        |
| 1102*                                                                                                                                      | 250       | 0         | 5480   | 0       | 4770 | -13    | -       | -    | -      |
| 1302                                                                                                                                       | 250       | 0         | 4340   | -       | -    | -      | -       | -    | -      |
| * Patient's treatment stopped due to study closure ADA data are from Roche "Bioanalytical Report (ADA) for Clinical Study BP29387"with ADA |           |           |        |         |      |        |         |      |        |  
* Patient's treatment stopped due to study closure ADA data are from Roche "Bioanalytical Report (ADA) for Clinical Study BP29387"with ADA
time point codes translated as specified in the BP29837 Lab Manual Version 1.0. Cmax data were taken from the Roche final study report.
untitled h1
1.2.4 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 (PDL2). 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.
untitled h1
1.2.4.1 pharmaceutical and therapeutic information

The importance of intact immune surveillance function in controlling outgrowth of neoplastic transformations has been known for decades.(15) 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 NSCLC, 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.(16, 17)
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-lymphocyte-associated 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).(18, 19) The structure of murine PD-1 has been resolved.(20) 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 tyrosinebased 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-chainassociated protein kinase (ZAP70), which are involved in the CD3 T-cell signaling cascade.(19, 21-23) 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.(24, 25) As a consequence, the PD-1/PD-L1 pathway is an attractive target for therapeutic intervention in NSCLC.
untitled h1
1.2.4.2 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.
untitled h1
1.2.4.2.1.2 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.B-17 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.(26, 27) A point mutation (S228P) in the core hinge region in IgG4 has been shown to be sufficient to prevent the Fab-arm exchange.(26, 27) The results supported that pembrolizumab, which has a hinge mutation from S to P at position 228, did not form detectable hybrid antibodies with coadministered 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.  
1.2.4.2.1.3 Assessment of Effects of PD-1 Blockade as Monotherapy and in Combination with Chemotherapy in Mouse Syngeneic Tumor Models
Anti-mouse 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. Anti-mouse 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 anti-mouse 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 anti-mouse 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 anti-mouse PD-1 J43 surrogate antibody in combination with gemcitabine also showed enhanced efficacy in the mouse MC38 colon adenocarcinoma tumor model.  
1.2.4.2.2 Clinical Investigations
untitled h1
1.2.4.2.2.1 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).(28-30) 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.(28) 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. 1.2.4.2.2.2 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 downmodulation 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, the Manufacturer Merck developed a broad list of immune-mediated adverse events of special interest (AEOSI) to evaluate and monitor. Additionally, though not immune-mediated, infusion-related reactions are also included in the AEOSI list. Based on ongoing monitoring, the manufacturer 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 Vogt-Koyanagi-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. 1.2.4.2.2.3 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 and do not have any sensitizing EGFR mutations or ALK
rearrangements. In the KEYNOTE-010 phase II/III trial, previously treated participants with PDL1 TPS ≥1% and disease progression following platinum-containing chemotherapy, pembrolizumab (2mg/kg and 10mg/kg) provided a statistically significant and clinically meaningful median OS benefit compared to standard docetaxel chemotherapy (10.4 and 12.7 months vs 8.5 months respectively) (31, 32). There was also a higher ORR (18% and 18% (pembro) vs 9% for docetaxel). For participants with previously untreated metastatic NSCLC whose tumors express high levels of PD-L1 (TPS ≥50%) and EGFR/ALKWT, the phase III KEYNOTE-024, pembrolizumab (200mg intravenous every 3 weeks) demonstrated significant improvements in the primary endpoint of PFS (10.3 vs 6 months) and OS (30 vs 14.2 months, preliminary results at 25-month follow up) over standard of care platinum-doublet chemotherapy. ORR was also significantly different for pembrolizumab vs platinum-doublet chemotherapy (45% vs 28%) as well as median duration of response (33). They phase III KEYNOTE-042 comparing pembrolizumab monotherapy with chemotherapy (TPS ≥1%) for untreated advanced NSCLC is currently ongoing. Pembrolizumab (200mg ever 3 weeks) in combination with pemetrexed/platinum agent for the frontline treatment of metastatic non-squamous NSCLC (EGFR/ALKWT) demonstrated both a statistically significant and clinically meaningful difference in OS, PFS, and ORR compared with chemotherapy alone in PD-L1 unselected patients. In the phase III KEYNOTE-189 OS (primary endpoint) was 69% vs 49% at 12 months for chemotherapy + pembrolizumab vs chemotherapy alone (34). Median PFS (co-primary endpoint) was 8.8 vs 4.9 months respectively as was ORR 48% vs 19%, respectively. The phase II (KEYNOTE-021) study led to the accelerated approval of chemotherapy (carboplatin, pemetrexed, and pembrolizumab) for metastatic, non-squamous NSCLC without activating *EGFR* mutations or ALK alterations in the frontline setting. 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 classical Hodgkin lymphoma (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 urothelial carcinoma (UC) in participants who have not received prior chemotherapy and are cisplatinineligible, 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.(35, 36)
untitled h1
1.2.5 rationale for combination therapy

Resistance to immunotherapy in NSCLC is a challenge and eventually results in disease progression. In an attempt to increase responses and overcome resistance to immune checkpoint inhibitors, combination therapy using an immunotoxin targeted to mesothelin expressing tumors may expand antigen spread and present new targets for immune recognition. Pre-clinical data show that combining immunotoxin with immune checkpoint blockade increases infiltration of CD8+ T cells and enhances anti-tumor immune response. We anticipate that LMB-100 will induce infiltration of CD8+ T cells and enhance anti-tumor immune response by expanding new tumor antigen targets for immune recognition. Primary resistance to immune checkpoint therapy in NSCLC is another challenge and can be tumor cell intrinsic driven from the absence of tumor specific antigenic proteins leading to ineffective Tcell rejection (37). LMB-100 immunotoxin combined with checkpoint blockade may overcome primary refractoriness to checkpoint inhibitors by initiating apoptotic cell death and releasing a broader repertoire of tumor specific proteins required to mount an anti-tumor response. 1.2.5.1 Combining local immunotoxin targeting mesothelin with CTLA-4 blockade synergistically eradicates murine cancer by promoting anti-cancer immunity. 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 LMB100) 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 (38). Surviving mice were protected from tumor re-challenge by 66C14 cells not expressing mesothelin, indicating the development of antitumor immunity directed to non-mesothelin tumor antigens. Although the mechanism is unknown, LMB-100 may be inducing antigen spread through apoptosis and priming immune cells to various other tumor specific antigens.  
Figure 2:**. LMB-100 and anti-CTLA-4 produces complete remissions but inactive LMB-100 (LMB100-I) does not.**  
  
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-PD1 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 intra peritoneally. 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 LMB-100 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.  
Abbreviated Title: LMB-100 plus pembro in NSCLC
Version Date: 12/3/2021 Figure 3. Combination therapy with LMB-100 plus anti-PD-1 antibody results in synergistic antitumor efficacy in hMSLN expressing mouse syngeneic lung cancer model
untitled h1
1.2.5.3 anti-tumor responses in patients

Table 4: A total of n= 9 patients with mesothelin expressing pleural or peritoneal mesothelioma were enrolled on trial at the NCI from August 2016- June 2018 and received immune-toxin LMB-100 with or without abraxane, followed by off-label pembrolizumab at progression. Results: two patients expired within 1-2 weeks of receiving pembrolizumab and were non-evaluable. The other seven patients were evaluable for response by RECIST. Prior treatments included cytoreductive surgery with HIPEC, chemotherapy with platinum-based combination with pemetrexed, navelbine, and anti-angiogenesis agent bevacizumab. Five of the seven patients received LMB-100 with abraxane, whereas two received LMB-100 alone. Overall responses to LMB-100 included three patients with stable disease (43%) and four patients (57%) with progressive disease. Progression free interval ranged from approximately 1-10 months. At progression, each patient went on to receive pembrolizumab off -label, each starting immunotherapy within one month upon progressing post LMB-100. Overall responses to pembrolizumab included four (57%) partial responders (range: 29.3%-67% decrease) and three progressive disease (43%). Progression free interval for responders ranged from 8 months to ongoing (>2 years), whereas for non-responders the interval was shorter, 2-4 months. A majority of non-responders (2/3) lacked PD-L1 IHC expression (unpublished data)  
| abraxane, whereas two received LMB-100 alone. Overall responses to LMB-100 included three patients with stable  disease (43%) and four patients (57%) with progressive disease. Progression free interval ranged from approximately  1-10 months. At progression, each patient went on to receive pembrolizumab off -label, each starting immunotherapy  within one month upon progressing post LMB-100. Overall responses to pembrolizumab included four (57%) partial  responders (range: 29.3%-67% decrease) and three progressive disease (43%). Progression free interval for responders  ranged from 8 months to ongoing (>2 years), whereas for non-responders the interval was shorter, 2-4 months. A  majority of non-responders (2/3) lacked PD-L1 IHC expression (unpublished data) Best Overall Time to Starting Progression Free Best Overall CT Patient Prior Treatments #LMB Cycles Progression Free Response to LMB- 100 Pembrolizumab after progression on LMB-100 Interval on Response to Interval on LMB-100 Pembrolizumab Pembrolizumab HIPEC, Cisplatin + Alimta x 6 4 10 months 17 days SD 1 month (33 days) 11 months, 14 days PR (-40%) 15%   |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 3+                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |
| (100%                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 |
| )                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
| 1                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
| Carbo                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 |
| +                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
| Alimta                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |
| x                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
| 9                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
| cycles                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |
| (2                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |
| w/                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |
| Avastin)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
| ,                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
| Maintenance                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           |
| Alimta                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |
| x                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
| 4,                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |
| Navelbine                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |
| x                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
| 4                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
| 2                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
| 1                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
| months,                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               |
| 17                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |
| days                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  |
| PD                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |
| <                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
| 1                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
| month                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 |
| (19                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |
| days)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 |
| Ongoing                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               |
| (>24                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  |
| months)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               |
| PR                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |
| (-64%)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |
| N/A                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |
| 3+                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |
| (95-100%)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |
| 2                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
| Cisplatin                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |
| +                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
| Alimta                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |
| x                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
| 2,                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |
| Carboplatin                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           |
| +                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
| Alimta                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |
| x                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
| 2,                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |
| HIPEC,                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |
| Carboplatin                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           |
| +                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
| 3                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
| Alimta                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |
| x                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
| 2,                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |
| HIPEC                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 |
| 2                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
| (w/                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |
| Abraxane)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |
| 5                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
| months,                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               |
| 15                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |
| days                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  |
| SD                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |
| <1                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |
| month                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 |
| (22                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |
| days)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 |
| 11                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |
| months,                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               |
| 10                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |
| days                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  |
| PR                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |
| (-29.3%)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
| 5-7%                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  |
| 2-3+                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  |
| (100%)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |  
  
  
epitheliod: 3+ (50-70% ) **sarcomatoid:** 0
untitled h1
1.2.6.1 lmb-100

The planned LMB-100 dose is the recommended single agent dose from the phase 1 study of LMB100 (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 most patients will develop neutralizing antibodies after 2 cycles of treatment. 1.2.6.2 Pembrolizumab For patients with metastatic non-squamous NSCLC, pembrolizumab at a dose of 200mg intravenously in combination with pemetrexed and carboplatin chemotherapy is FDA approved as a front-line therapy irrespective of (PD-L1) expression. In the phase III KEYNOTE- Gandhi et al (34) demonstrated that overall responses was improved with the addition of pembrolizumab 48% versus 19%. Pembrolizumab combined with chemotherapy also improved 12-month OS rates relative to chemotherapy alone 69 versus 49%, and median PFS 8.8 vs 4.9 months across all PDL1 categories.  
Pembrolizumab is also approved in the frontline setting whose tumors have ≥50% PD-L expression. Reck et al demonstrated that in the phase III KEYNOTE-024 trial that untreated patients with metastatic NSCLC having at least 50% tumor cell PD-L1 expression, ORR was improved with pembrolizumab compared to platinum-doublet chemotherapy 45 vs 28% (33).  
Median PFS (the primary endpoint), was also increased 10.3 versus 6 months. 25-month followup demonstrate improved median OS with pembrolizumab 30 versus 14.2 months (39).  
Finally, pembrolizumab 200mg IV every three weeks demonstrates activity and survival benefit in patients who have been treated with platinum-based chemotherapy and is approved for tumors with at least 1% PD-L1 expression. Herbst et al randomized patients with metastatic NSCLC and at least 1% PD-L1 TPS to pembrolizumab or chemotherapy with docetaxel in the phase II/III KEYNOTE-10 trial (31). Median OS was improved with pembrolizumab of 12.7 months (higher dose) vs 8.5 months for docetaxel. ORRs were better in the pembrolizumab versus chemotherapy, 18 vs 9%. 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 participant 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 exposure-response 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. 1.2.6.3 Combination treatment There is no phase I data of this combination of LMB-100 and pembrolizumab. However, the combination will be given sequentially with LMB-100 for two cycles followed by pembrolizumab rather than concurrently. We do not anticipate overlapping toxicities as the side effects profiles of LMB-100 (capillary leak syndrome, weight gain, atrial fibrillation) and pembrolizumab (immune related events) are different. In addition, treatment with pembrolizumab will not be initiated until LMB-100 related toxicities have resolved to grade 1 or better. Currently, we have a study in mesothelioma of LMB-100 followed by pembrolizumab (18C0136C). Of the 5 patients who completed two cycles of LMB-100, four have received two doses of pembrolizumab without any unanticipated toxicities.  
We do have experience with patients who received LMB100 who received prior checkpoint therapy. In a prior study of LMB-100 in mesothelioma patients (16-C-0127), five patients received pembrolizumab prior to receiving LMB-100. None of these five patients unanticipated toxicities from receiving LMB-100 following immune checkpoint inhibitor. 1.2.7 Hypotheses and Summary We believe that anti-mesothelin immunotoxin LMB-100 followed by pembrolizumab will result in anti-tumor efficacy in subjects with non-squamous, NSCLC by augmenting antitumor immunity.  
We hypothesize that LMB-100 will induce tumor direct inflammation that leads to recruitment of CD8+ cells in the tumor and administration of pembrolizumab will increase the efficacy of these cytotoxic T cells.
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 (38). Based on clinical trials of pembrolizumab therapy in subjects with refractory NSCLC which typically demonstrate responses of 18-20%, we propose a clinical trial of this combination to determine if administration of LMB-100 followed by pembrolizumab can induce anti-tumor immunity and achieve a desired response rate of 10% or greater in subjects who have previously failed a checkpoint inhibitor.
untitled h1
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 non-squamous non-small cell lung cancer not amenable to potentially curative treatments (surgical resection, definitive radiation therapy or a combined modality approach) or targeted agents to actionable EGFR mutations or ALK or ROS1 gene rearrangement and excluding neuroendocrine tumors. Activating KRAS mutations are allowed. The diagnosis must be confirmed by the Laboratory of Pathology, CCR, NCI. Mutation confirmation may be done by referring institutions or by one of the assays in section **2.2.2.** 2.1.1.3 Have provided archival tumor tissue sample or newly obtained fresh 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. 2.1.1.4 Histologically confirmed 25% of tumor cells expressing mesothelin as determined by NCI Laboratory of Pathology. Determination can be made using archival tumor tissue or fresh biopsy. 2.1.1.5 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. 2.1.1.6 Subjects must have received prior standard of care treatments for locally advanced or metastatic NSCLC. 2.1.1.7 Patients must be more than 3 weeks out of systemic treatments, such as chemotherapy. 2.1.1.8 All acute toxic effects of any prior radiotherapy, chemotherapy, immunotherapy, or surgical procedure must have resolved to Grade less than or equal to 1, except alopecia (any grade) and Grade 2 peripheral neuropathy. 2.1.1.9 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. 2.1.1.10 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                                                                                      | ≤1.5 × ULN OR                                                                                                |                                                                               |             |          |           |                |
| -                                                                                                          | Measured                                                                                                     | or                                                                            | calculatedb |          |           |                |
| creatinine clearance (GFR  can also be used in place of  creatinine or CrCl)                               | ≥ 50 mL/min for participant with creatinine levels >  1.5 X institutional ULN                                |                                                                               |             |          |           |                |
| Coagulation - International                                                                                | normalized                                                                                                   |                                                                               |             |          |           |                |
| ratio                                                                                                      | (INR)                                                                                                        | OR                                                                            |             |          |           |                |
| prothrombin time (PT)                                                                                      | ≤1.5 × ULN                                                                                                   | unless                                                                        | participant | is       | receiving |                |
| anticoagulant therapy as long as PT or aPTT is within  therapeutic range of intended use of anticoagulants |                                                                                                              |                                                                               |             |          |           |                |
| -                                                                                                          | Activated                                                                                                    | partial                                                                       |             |          |           |                |
| thromboplastin time (aPTT)                                                                                 |                                                                                                              |                                                                               |             |          |           |                |
| ALT                                                                                                        | (SGPT)=alanine                                                                                               | aminotransferase                                                              | (serum      | glutamic | pyruvic   | transaminase); |  
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.  
2.1.1.11 Must have left ventricular ejection fraction >50%. 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 Ability of subject to understand and the willingness to sign a written informed consent document. 2.1.1.14 Subjects with non-life-threatening immune-related endocrinopathies or AEs reduced to Grade 1 or 0 after withholding ICI or medical intervention are eligible as long as the AE resolved within 12 weeks of last dose and not requiring corticosteroids.
untitled h1
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 Subjects who have received prior therapy with LMB-100. Has received prior systemic anti-cancer therapy including investigational agents within 4 weeks prior to start of study therapy.  
Note: Participants must have recovered from all AEs due to previous therapies to ≤Grade 1 or baseline. Participants with ≤Grade 2 neuropathy may be eligible. Note: If participant received major surgery, they must have recovered adequately from the toxicity and/or complications from the intervention prior to starting study treatment.  
2.1.2.4 Has received prior radiotherapy within 2 weeks of start of study treatment. Participants must have recovered from all radiation-related toxicities, not require corticosteroids, and not have had radiation pneumonitis. A 2-week washout is permitted for palliative radiation (≤2 weeks of radiotherapy) to non-CNS disease. 2.1.2.5 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. 2.1.2.6 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. 2.1.2.7 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.  
2.1.2.8 Has a history of (non-infectious) pneumonitis/interstitial lung disease (ILD) that required steroids or has current pneumonitis/ILD 2.1.2.9 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. 2.1.2.10 Has known psychiatric or substance abuse disorders that would interfere with cooperation with the requirements of the trial. 2.1.2.11 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. 2.1.2.12 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. 2.1.2.13 Has a known history of Human Immunodeficiency Virus (HIV). 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) 2.1.2.14 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.) 2.1.2.15 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. 2.1.2.16 Has an active infection requiring systemic therapy. 2.1.2.17 Participants with contra-indication and/or history of severe hypersensitivity reactions to any components related to LMB-100 or pembrolizumab (or any other immune checkpoint inhibitor such as PD1, PDL-1 and CTLA4). 2.1.2.18 Active or uncontrolled infections. 2.1.2.19 Subjects who experienced severe or life-threatening immune-related AEs with prior immune checkpoint therapy requiring medical intervention (steroid or immunosuppressant drugs) and permanent discontinuation of therapy, will be excluded. These include, but not limited to colitis, autoimmune hepatitis, hypophysitis, hyperthyroidism, nephritis, myocarditis, GBS, encephalitis. 2.1.2.20 Subjects with a history of pneumonitis that required steroids will be excluded.
untitled h1
2.1.3 recruitment strategies

Information about the study will be posted on sites such as clinicaltrials.gov and the CCR recruitment website. Subjects will also be drawn from patients seen at the thoracic clinic at the NIH Clinical Center as well as from referrals from outside providers. Social media platforms managed by NIH/NCI may also be used to publicize the study. There is no plan to advertise this study at this time.