Patent ID: 12247078

The DOC4-NRG1 and CLU-NRG1 gene fusions are expressed in the MDA-MB-175 cell line (breast) and in the OV-10-0050 PDX (ovarian), respectively. Left panel: in vitro MCLA-128 treatment inhibits MDA-MB-175 cell proliferation. Right panel: In vivo, MCLA-128 treatment (25 mg/kg weekly until day 28) reduced tumor growth and eliminated tumors in 6/8 animals.

EXAMPLES

Example 1: ErbB-2-Guided Targeting

An imaging experiment was performed comparing the HER2×HER3 bispecific antibody (PB4188) to the HER3 bivalent monoclonal antibody. Variants of bAb PB4188 and anti-HER3 MF3178 (parental antibody) were labelled with 64Cu and injected intravenously in mouse xenografted with HER2 gene-amplified JIMT-1 tumors. Micro-PET imaging demonstrated that the PB4188 variant more effectively accumulated in tumors compared to the HER3 monoclonal (FIG.2A).

Gamma-counter quantification of radioactivity present in tumors confirmed that levels of PB4188 variant in the tumors were 2.5-fold higher than for the parental anti-HER3 antibody (FIG.2B). Overall, in vitro and in vivo data demonstrate that HER2-targeting is responsible for enhanced binding of PB4188 on tumor cells. Additional studies were performed using an anti-HER2 (MF3958) antibody.FIG.2Csummarizes the results of the respective antibodies labelled with 64Cu and injected in mouse xenografted with HER2 gene-amplified JIMT-1 tumors (n=4 mice for each antibody treatment).

Methods

Biodistribution study. Variants of bAb PB4188, anti-HER2 MF3958, and anti-HER3 MF3178 were conjugated to a bifunctional chelator [Paterson 2014 Dalton Transactions]. Binding characteristics of the conjugated products to the target were confirmed using flow cytometry-based assays. Proteins were then labelled with 64Cu and mice bearing JIMT-1 breast xenografts were administered the radiolabeled antibodies via tail vein (FIGS.2A-Band “i.v.” forFIG.2C) or intraperitoneal (“i.p.” forFIG.2C). MicroPET/CT images were acquired 48 hrs post-injection, after which tumor were excised and radioactivity was measured in a gamma counter. Results were expressed as percentage injected dose per gram tissue.

Example 2 Inhibition of Heterodimer Formation

Heterodimerization assays based on the enzyme fragment complementation technology were used. The 6-galactosidase enzyme can be artificially split into two inactive fragments, the enzyme donor and the enzyme acceptor, which combine into an active enzyme only when in close proximity. Each sequence encoding either the enzyme donor or the enzyme acceptor is linked to the extracellular and transmembrane domains of each heterodimerization partner. Both genes are then co-transfected in U2OS cells to express extracellular domains of RTK receptors linked to one domain of 6-galactosidase (ED or EA). Upon agonistic stimulation of one RTK receptor, both RTK receptors dimerize, inducing formation of an active fully reconstituted 6-galactosidase enzyme. Ultimately, 6-galactosidase activity is measured by adding a substrate that upon hydrolyzation will lead to light emission.

Antibodies where tested in EGFR:HER2, HER2:HER3 and HER3:HER4 heterodimerization reporter cell lines. RTK heterodimerization assays were run with the bispecific antibody MCLA-128 (MF3178 arm and MF3958 arm); anti-HER3 antibodies MF3178/PG3178 and PG3793/AMG-888/patritumab; and anti-HER2 antibodies MF3958/PG3958, PG2867/trastuzumab, PG2869/pertuzumab, and Perjeta (clinical batch of pertuzumab). EGF and HRG titrations in EGFR:HER2 and HER2:HER3, HER2:HER4 assays showed dose-dependent agonist responses (FIG.3). MCLA-128 showed complete inhibition of HER2:HER3 dimer formation specifically and had no effect on EGFR:HER2 or HER2:HER4 heterodimerization. In contrast, trastuzumab (PG2867) behaved as partial antagonist in EGFR:HER2 and HER2:HER3 assays.

MCLA-128 and PG3178 fully inhibited HRG-induced HER2:HER3 dimerization with the highest potency (Table 1).

TABLE 1Summary results of EC50 of antibodies tested in RTKheterodimerization assays. EC50 were determined non-linear regressions (4 parameters) in Prism.IC50 (nM)EGFR:HER2HER2:HER3HER2:HER4PG1337———PB4188—1.12—PG3187—1.27—PG3958——1.69PG28670.304.910.63PG28690.474.222.91PG3793—3.23—Perjeta0.616.265.44Agonist0.020.220.08

The potency of trastuzumab was about 4-fold lower than MCLA-128 or PG3178 in the HER2:HER3 assay. Perjeta (clinical pertuzumab) behaved as full antagonist in all three assays and gave a similar profile as PG2867 (pertuzumab). In HER2:HER4 assays, both anti-HER2 PG3958 and PG2867 (pertuzumab) showed minor decreases in dimerization that appeared to be dose-dependent. Small non-specific responses in EGFR:HER2 assays were observed at high concentrations of PG1337, MCLA-128, PG3178 and PG3958.

MCLA-128 showed specific inhibition of HER2:HER3 heterodimers only. This indicates that upon binding on HER2, MCLA-128 should not sterically impair interaction of HER2 with EGFR upon EGF stimulation, nor impair heterodimerization of HER2 with HER4 upon HRG stimulation.

The latter is in line with observation in the HRG-induced cell cycle-based proliferation assay of T47D cells. Assays using these cells failed to demonstrate inhibitory activity of MCLA-128 or PG3178, which was presumably attributed to the higher expression of HER4 compared to HER3. HRG is thought to preferably signal via HER2:HER4 in T47D cells instead of HER2:HER3, explaining the lack of efficacy of MCLA-128 and indicating a specificity of MCLA-128 for HRG-induced HER2:HER3 dimers and not for HRG-induced HER2:HER4 dimers.

In the current study, trastuzumab blocked EGF- and HRG-induced heterodimerization of EGFR:HER2 and HER2:HER3, respectively. Trastuzumab and pertuzumab behaved as partial and full antagonist, respectively, which is in line with the generally accepted claim that trastuzumab blocks ligand-independent activation of HER2 while pertuzumab inhibits ligand-dependent signaling. The fact that a trastuzumab inhibitory response is observed in these assays might be due to the overexpression of both targets. This might allow a more sensitive readout than traditional immunoprecipitation experiments.

Finally, while PG3793 showed a lower binding affinity than PG3178 on MCF-7, its lower potency in HER2:HER3 heterodimerization assay is less severe (2.5-fold difference in dimerization assay potency versus 30-fold difference in binding assay affinity). This discrepancy between binding affinity and antagonism potency has previously been observed in the case of MCLA-128 and PG3178. While PG3178 binds MCF-7 with a slightly better affinity than MCLA-128, MCLA-128 outperforms PG3178 in a cell cycle-based proliferation assay.

Example 3

Study Objective and Regulatory Compliance

The objective of the research is to evaluate the in vivo anti-tumor efficacy of MCLA-128, PG2863 and PG2869 antibodies in the treatment of the subcutaneous human ovarian cancer PDX model of OV-10-0050 in BALB/c nude mice.

Experimental Design

The experimental design is indicate in table 2. In all groups, blood was sampled in 4 animals on day 2 (24 hr post first dose) and in the remaining 4 animals on day 6 (5 days post first dose). At the designated time point, 50-100 μl blood was collected into sterile collection tubes (Microvette CB300Z clotting activator/serum, Sarstedt B.V. cat #16.440.100), allowing the samples to clot at room temperature for 45 minutes, centrifuging for 10 minutes at 3000 rpm and taking up the aqueous layer (about 20 μl serum) into another 1.5 mL sterile Eppendorf for immediate storage at −80° C. Samples are shipped on dry ice.

TABLE 1Description of Experimental DesignDoseDosevolumeConc.GroupNaTreatmentmg/kgml/kgbmg/ml18Vehicle Control—10—(DPBS)28MCLA-12825102.538PG286325102.548PG286925102.5aN: number of animals per group;bDose volume: adjust dosing volume based on body weight 10 μl/g.

All animals were treated on day 1, 8, 15, 22, 29 (weekly treatment for 5 weeks) Route was I.P. for all groups

Tumor samples were harvested at 48 hr post last dose (day 31). Tumors were fixed in neutral buffered formalin (tissue:fixative ratio of at least 1:20) for 24 hrs, and then converted to FFPE blocks.

Preparation of Neutral buffer formalin: Put one bag of PBS powder in a clear 5 L-volumetric flask, added 4.5 L de-ionized water and stirred to disperse the powder to obtain a clear solution. Then added 500 ml formaldehyde to stir until a homogenous solution was achieved.

Materials

Animals: Species:Mus musculus: Strain: BALB/c nude; Age: 6-8 weeks; Sex: female Body weight: 18-22 g; Number of animals: 32 mice plus spare Animal supplier: Shanghai Sino-British SIPPR/BK Laboratory Animal Co., LTD.

Diet: Animals had free access to irradiation sterilized dry granule food during the entire study period; Water: Animals had free access to sterile drinking water.

Antibody Package and Storage Condition:

MCLA-128; cryovials, 10×1.5 ml/vial at 2.5 mg/ml, stored at 4° C.

PG2863; cryovials, 10×1.5 ml/vial at 2.5 mg/ml, stored at 4° C.

PG2869; cryovials, 10×1.5 ml/vial at 2.5 mg/ml, stored at 4° C.

Generation of the PDX Model

The human ovarian cancer PDX model of OV-10-0050 was originally established from a 48 years old female patient presenting grade 3 adenocarcinoma of the ovaries. A surgically resected clinical sample was implanted in nude mice (defined as passage 0, P0) and the following serial implantations were defined as P1, P2, etc. The P6 tumor tissue was used for this study.

Tumor Implantation Each mouse was implanted subcutaneously at the right flank with the OV-10-0050 P6 tumor slices which cut by scissor (˜30 mm3) for tumor development. The treatments were started on day 30 after tumor implantation when the average tumor size reached approximately 152 mm3. 32 tumor bearing mice were randomized into 4 groups with a stratified randomization method and each group consisted of 8 tumor-bearing mice. The day of randomization was noted as day 1, and it was day of start of treatment. The test articles were administered to the mice according to the predetermined regimen as shown in the experimental design table (Table 2).

Observations

All the procedures related to animal handling, care and the treatment in the study were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi AppTec following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). At the time of routine monitoring, the animals were daily checked for any effects of tumor growth and treatments on normal behavior such as mobility, food and water consumption (only by visual inspection), body weight gain/loss (body weights were measured twice weekly), eye/hair matting and any other abnormal effect as stated in the protocol. Death and observed clinical signs were recorded on the basis of the numbers of animals within each subset.

Tumor MEASUREMENTS

Tested was whether the tumor growth could be delayed or if mice could be cured. Tumor size was measured twice weekly in two dimensions using a caliper, and the volume was expressed in mm3 using the formula: V=0.5 a×b2 where a and b are the long and short diameters of the tumor, respectively. The tumor size was then used for calculations of T-C, T/C and TGI values. T-C was calculated with T as the median time (in days) required for the treatment group tumors to reach a predetermined size (e.g., 500 mm3), and C as the median time (in days) for the control group tumors to reach the same size. The T/C value (in percent) is an indication of antitumor effectiveness; T and C were the mean volumes of the treated and control groups, respectively, on a given day. TGI was calculated for each group using the formula: TGI (%) [1−(Ti−T0)/(Vi−V0)]×100; Ti was the average tumor volume of a treatment group on a given day, T0 was the average tumor volume of the treatment group on the first day of treatment, Vi was the average tumor volume of the vehicle control group on the same day with Ti, and V0 was the average tumor volume of the vehicle group on the first day of treatment.

Statistical Analysis

Summary statistics, including mean and the standard error of the mean (SEM), are provided for the tumor volume of each group at each time point (detailed in table 3). Statistical analysis of difference in tumor volume among the groups and the analysis of drug interaction were conducted on the data obtained at the best therapeutic time point after the final dose (the 29th day after grouping).

A one-way ANOVA was performed to compare tumor volume among groups, and when a significant F-statistics (P<0.001, a ratio of treatment variance to the error variance) was obtained, comparisons between groups were carried out with Games-Howell. All data were analyzed using SPSS 17.0. p<0.05 was considered to be statistically significant.

Results

Mortality, Morbidity, and Body Weight Gain or Loss

Animal body weight was monitored regularly as an indirect measure of toxicity. No groups lost weight as a result of test article administration (FIG.4) and no deaths or morbidity were observed. Thus, there does not appear to be obvious toxicity associated with administering MCLA-128, PG2863 and PG2869 antibodies to tumor-bearing BALB/c nude mice.

Body weight change in female BALB/c nude mice bearing OV-10-0050 xenografts dosed with MCLA-128, PG2863 and PG2869 antibodies are shown inFIG.4andFIG.5. Mean tumor volume over time in female BALB/c nude mice bearing OV-10-0050 xenografts dosed with MCLA-128, PG2863 and PG2869 antibodies are shown in Table 3.FIG.6shows the tumor growth.

TABLE 3Tumor volume over time (mm3)aVehicleMCLA-128PG2863PG2869Daysb(DPBS)25 mg/kg25 mg/kg25 mg/kg1152 ± 17151 ± 21152±152 ± 215239 ± 1875 ± 1287 ± 1041 ± 88332 ± 2669 ± 15115±12 ± 112434 ± 5047 ± 1198 ± 139 ± 115520 ± 6336 ± 10108±6 ± 119598 ± 6426 ± 892 ± 164 ± 122718 ± 8823 ± 8106±3 ± 126911 ± 11821 ± 991 ± 162 ± 1291,161±23 ± 11108±1 ± 0aMean ± SEM;bStudy day
Results and Discussion

In the study, the therapeutic efficacy of MCLA-128, PG2863 and PG2869 antibodies as a single agent in the treatment of the OV-10-0050 human ovarian cancer xenograft model was evaluated. The results of tumor sizes in different groups at different time points after tumor inoculation are shown in the Table 3, Table 4, andFIG.4.

The mean tumor size of the vehicle treated control mice reached 1,161 mm3at day 29 after grouping. Treatment with the test articles MCLA-128, PG2863 and PG2869 antibodies at 25 mg/kg (QW×5 weeks) produced significant antitumor activity: their mean tumor sizes were 23, 108 and 1 mm3, respectively at the same time (T/C value=1.95%, 9.28% and 0.06%; TGI value=112.78%, 104.37% and 114.96%; p value=0.002, 0.003 and 0.002, respectively) their tumor growth all delays more than 14 days, at the tumor size of 500 mm3compared with the vehicle group. Treatment causes partial regression or complete regression of the tumor. A mouse was considered to have a partial regression (PR) when tumor volume was reduced 50% or greater of its day 1 volume for three consecutive measurement during the course of the study and >13.5 mm3for one or more of these three measurements. And to have a complete regression (CR) when <13.5 mm3for three consecutive measurement during the course of the study. Tumor free survival was considered as no palpable tumor was detected at the end of the study.

Treatment with MCLA-128, PG2863 and PG-2869 resulted different ratio of PR, CR and TFS. The mouse amount in each group that shows PR, CR and TFS is shown is table 5. All the testing articles were tolerated well by the tumor-bearing animals. No body weight loss was observed in all of treatment groups.

In summary, the three test antibodies as single agent all produced significant antitumor activity against the OV-10-0050 human ovarian cancer xenograft model in this study. It was well tolerated by the tumor-bearing animals. The results indicated that the antibodies are safe and effective anticancer agents.

TABLE 4Tumor growth inhibition calculation for injected MCLA-128; PG2863and PG2869 antibodies in the OV-10-0050 model calculated basedon tumor volume measurements at day 29 after grouping.Tumor SizeT/CbTGIT-C (days) AtpTreatment(mm3)a(%)(%)500 mm3valuecVehicle (DPBS)1,161 ± 182—0—MCLA-128 (25 mg/kg)23 ± 111.95112.78>140.002PG2863 (25 mg/kg)108 ± 229.28104.37>140.003PG2869 (25 mg/kg)1 ± 00.06114.96>140.002aMean ± SEM.bTumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C). For a test article to be considered to have anti-tumor activity, T/C must be 50% or less. TGI is calculated using the formula TGI (%) = [1 − (Ti − T0)/(Vi − V0)] × 100.cp value is calculated based on tumor size, compared with the vehicle group.

TABLE 5PR, CR and TFS statistics for different treatmentsTreatmentPRCRTFSMCLA-128 (25 mg/kg)350PG2863 (25 mg/kg)300PG2869 (25 mg/kg)083

Example 4

MCLA-128 is a bispecific antibody targeting HER2 and HER3 receptor tyrosine kinases (RTK), which are involved in the proliferation and survival of cancer cells. MCLA-128 has been extensively studied in the context of heregulin (HRG)-induced HER3 signaling and proliferation. It has demonstrated stronger in vitro potency than: the combination of anti-HER2 antibodies pertuzumab (PG2869)+trastuzumab (PG2867), which can block ligand-dependent and ligand-independent HER2:HER3 signaling, respectively [Agus 2002; Juntilla 2009]; the anti-HER3 MM-121 (PG2863), which blocks HRG-induced HER3 activation [Schoeberl 2010].

MCLA-128 also shows anti-tumor activity in cells expressing gene fusions involving the HRG gene. The MDA-MB-175 cell line contains the DOC4-NRG1 gene fusion, which results in a proliferative autocrine loop due to NRG1 expression. This gene fusion has until this date not been discovered in a cancer patient setting [Sanchez-Valdivieso 2002].

From a panel of breast cancer cell lines, MDA-MB-175 cells were sensitive to single agent MCLA-128, demonstrating the importance of the HER3/HRG signaling axis in this cell line (FIG.7left panel). The activation of the HER2 in this cell line has also been demonstrated in vivo, where a single dose of pertuzumab, but not trastuzumab, inhibited orthotopic MDA-MB-175 tumor growth. Although the relevance of DOC4-NRG1 gene fusion in breast cancer patients has been debated [Sanchez-Valdivieso 2002], other gene fusions have recently gained interest. In particular, the CD74-NRG1 fusion has been reported by independent groups in invasive mucinous adenocarcinoma, a subpopulation of non-small cell lung cancer [Fernandez-Cuesta 2014, Duruisseaux 2016]. Several other NRG1 gene fusions have also been detected, namely VAMP2-NRG1, RBPMS-NRG1 and WRN-NRG1 in lung cancer, as well as RAB2IL1-NRG1 in ovarian cancer [Jung 2015, Dhanasekaran, 2014]. This diversity of gene fusions may be related to the location of NRG1 gene on chromosome 8, which is susceptible to translocations [Adelaide 2003].

OV-10-0050 was found to be HER-dependent. Treatment with afatinib (an irreversible inhibitor of EGFR and HER2 that also inhibits transphosporylation of HER3) led to tumor growth inhibition. The anti-tumor efficacy of MCLA-128 was compared to PBS (FIG.7, right panel).

Mice: NOD-SCID, Crl:NU(NCr)-Foxn1nu and BALB/c Nude mice. Antibodies are dosed at 25 mg/kg during 4 weeks. Tumor volumes are measured by caliper twice a week.

Example 5: A Phase I/II Study of MCLA-128, a Full Length IgG1 Bispecific Antibody Targeting HER2 and HER3, in Patients with Solid Tumors

Study Duration:

Accrual to the dose escalation part of the study (Part 1, first patient dosed on Feb. 3, 2015), has been completed after recruiting 28 patients. The first patient in Part 2 of the study, the dose expansion phase, was dosed on 15 Jan. 2016 in Europe. The total duration of Part 2 is approximately 25-32 months; however, the actual duration is influenced by several variables, e.g., overall subject recruitment rate.

Number of Sites:

Up to 13 sites are estimated to be involved during the study. Additional sites may be added to ensure there is an acceptable enrollment rate or to replace non-enrolling/withdrawn sites.

Number of Patients:

Twenty-eight (28) patients were enrolled in Part 1. For Part 2, at least 20 evaluable patients, and up to approximately 40, may be enrolled in the grouped advanced/metastatic non-small cell lung cancer with invasive mucinous adenocarcinoma or documented NRG1 fusion; NSCLC).

Patients who do not complete at least two cycles of study treatment due to other reasons than disease progression, are not evaluable for efficacy and are replaced in the respective group.

This Example describes Part 2. While the example describes the administration of MCLA-128, an Erb-2, Erb-3 binding bispecific antibody, the example is not intended to be limiting to the use of this specific embodiment and applies to other bispecific antibodies disclosed herein.

Study Objectives:

Part 1

ObjectivePrimary:Determination of the MTD and/or MRDEvaluation of adverseof MCLA-128.events (AEs) and doselimiting toxicities (DLT).Secondary:To characterize the safety andFrequency and nature oftolerability of MCLA-128.AEs/serious adverse events(SAEs).PK profile of MCLA-128.Assessment of PK variables,including total exposure,maximum concentration(Cmax) clearance, volume ofdistribution (V), volume ofdistribution at steady state(Vss), half-life (t1/2), AUC0-t(area under theconcentration versus timecurve from time zero to timet), AUC0-∞(area under theconcentration versus timecurve), tmax(time to reachmaximum concentration).Immunogenicity of MCLA-128.Incidence and serum titersof anti-drug antibodiesagainst MCLA-128.Evaluation of anti-tumorAnti-tumor activity andresponse and CBR.clinical benefit assessed byRECIST v1.1 determiningobjective overall responserate (ORR), duration ofresponse (DOR),progression-free survival(PFS) and survival; CBR isdefined as the proportion ofpatients in whom acomplete response (CR) orpartial response (PR) orstable disease (SD) isobserved (where SD)duration is a minimum of12 weeks).Exploratory (includes optionalassessments):Presence of biomarkers andAssessment of relevantpharmacodynamic (PD)tumor biomarkers andresponses to MCLA-128.markers of MCLA-128activity in archival and/orfresh tumor biopsy materialand blood. The followingcandidate biomarkers areassessed:HER2, HER3, pHER2,pHER3 & heregulin;KRAS, NRAS, PIK3CA,BRAF mutation status(metastatic colorectalcancer (mCRC) patientsonly);circulating tumordeoxyribonucleic acid(DNA) and mutations ingenes associated withHER2/HER3 signaling;phosphorylatedmolecules in the MAPKand AKT signalingpathway.
Part 2

ObjectivePrimary (safety):To characterize the safety andFrequency and nature oftolerability of MCLA-128.AEs.Primary (efficacy):To explore the relationships between theOverall response rateanti-tumor activity of MCLA-128 and(ORR), DOR, CBRdisease-related biomarkers(defined as theproportion of patients inwhom a CR or PR isobserved, or SD of aminimum duration of 12weeks) per RECIST 1.1as per localinvestigator'sassessment. Therelationship betweenanti-tumor activity andbiomarkers includingexpression of HER2,HER3, and heregulinare explored, and serumbiomarkers such as CA-125 (ovarian,endometrial) and CA-19-9 (gastric)Secondary:PK profile of MCLA-128.Assessment of PKvariables, including totalexposure, Cmax, V, Vss, t1/2,AUC0-t, AUC0-∞, tmax.Population PK analysisImmunogenicity of MCLA-128.Incidence and serum titersof anti-drug antibodiesagainst MCLA-128.Evaluation of PFS and overall survival,duration of responseExploratory (includes optional assessments):Assessment of other relevant tumorThe following candidatebiomarkers and markers of MCLA-128biomarkers are assessed ifactivity in preferably fresh tumorsufficient sample issample/biopsy material or archival andavailable:blood.Tumor samplepHER2, pHER3,HER2:HER3dimerization;Heregulin and(depending onavailability) mutationsin cancer genesincluding thoseassociated with HER2and HER3Phosphorylatedmolecules in the MAPKand AKT signalingpathway.Heregulin-gene fusionsBloodFcgamma receptorpolymorphismCirculating tumor DNAand mutation analysisin cancer genesincluding thoseassociated withHER2/HER3 signaling;Circulating tumor cellsand HER2 status
Study Design:

This is a Phase I/II, open-label, multi-center, multi-national, dose escalation, single group assignment study to assess the safety, tolerability, PK, PD, immunogenicity and anti-tumor activity of MCLA-128.

The study is designed in 2 parts:

Part 1

Accrual to Part 1 of the study was met on 24 Nov. 2015 and as of 24 Jan. 2017 all Part 1 patients had completed the study. Nine dose levels were investigated: 40 mg, 80 mg, 160 mg in cohorts of 1 patient and 240 mg, 360 mg, 480 mg, 600 mg, 750 mg, and 900 mg in cohorts of 3 patients. MCLA-128 was initially given over approximately 60 minutes on Day 1 of a 3-week treatment cycle. During Part 1 the infusion duration was extended to 2 hours with the option of increasing it up to 4 hours to mitigate infusion-related reactions (IRRs).

No dose limiting toxicities (DLTs) were experienced at any of the dose levels. Three additional patients were dosed in each of the 600 mg and 750 mg cohorts in order to have sufficient PK information.

As an MTD was not reached at the dose level of 900 mg, the Data Review Committee (DRC) for MCLA-128-CL01 decided to assign the dose level of 750 mg as the RP2D of the study, based on the cumulative safety, available PK data and PK simulations.

Part 2

Part 2 includes a further characterization of the safety and tolerability of the selected dose level of MCLA-128, as well as assessment of CBR, defined as the proportion of patients with a CR, PR or durable SD (SD for at least 12 weeks in duration), in expansion groups of selected patient populations.

A weekly dose regimen with a 4-week cycle is evaluated in newly recruited patients consisting of a flat dose of 400 mg weekly for the first 2 cycles, with an 800 mg loading dose for the initial administration. From cycle 3, MCLA-128 is given at a dose of 400 mg weekly for 3 weeks followed by 1 week off. Mandatory pre-medication is administered to mitigate IRRs. However, corticosteroids are only mandatory prior to the loading dose of Day 1 of Cycle 1 and should only be used for subsequent infusions as per the investigator's discretion to manage IRRs.

Safety of the weekly schedule is reviewed during a run-in period after the first 5 patients treated have completed at least 2 treatment cycles. The DRC reviews all safety data with a focus on incidence of grade 3-4 toxicities, incidence and severity of IRRs, and compliance. If the DRC review concludes that toxicity is unacceptable, the Sponsor continues patient enrolment with the 3-week cycle dose regimen until a sufficient number of patients have been enrolled per cohort.

No within-patient dose escalation is permitted in Part 2.

Patient populations of interest to be assessed in Part 2 of the study are:NSCLC with documented NRG1 fusions-only open for recruitment in Asia

At least 20 and up to approximately 40 patients may be enrolled in each Group (C-F) including a minimum of 10 patients per cohort treated with the weekly recommended dose. Previously closed cohorts may be reopened.

Duration of Treatment

Patients in both Part 1 and 2 of the study may remain on treatment until disease progression, death, unacceptable toxicity or discontinuation for any other reason.

Data Review Committee (DRC):

All dose escalation decisions in Part 1 were made by a DRC who convened to review all available safety data and PK data. The DRC participants included the Principal Investigators (or their representatives), the Sponsor's Medical Director, the study Medical Monitor, study Pharmacovigilance Physician, study Project Manager, study Statistician, and invited experts as required (such as clinical pharmacology expert).

In Part 2, the DRC reviews the data following completion of the safety run-in period for the weekly dose before expanding the weekly dose regimen in all subsequent patients.

Study Assessments:

The study consists of a molecular pre-screening assessment up to a 4-week (28-day) screening period, followed by sequential treatment cycles until treatment withdrawal or termination for any reason. The treatment cycle duration is 3 weeks (21 days) for patients treated at the initial recommended dose in Part 2, and 4 weeks (28 days) for patients treated at the weekly recommended dose in Part 2. All patients should attend an End of Treatment visit within 1 week after treatment cessation and a Final Study Visit 30 days after end of treatment or discontinuation from study.

Patients who have not progressed or withdrawn consent on completion of the Final Study Visit are followed up every 3 months for up to 2 years (approximately) to check their disease progression and/or survival status until the commencement of their next anti-cancer treatment.

Where ongoing evaluation of safety data and available PK, PD and anti-tumor activity data during the trial suggest that alternative dosing frequencies should be evaluated, or that other patient populations should be evaluated in Part 2, these modifications are clarified in a protocol amendment prior to commencing these evaluations.

Molecular Pre-Screening and Screening:

Molecular pre-screening is performed in local laboratories qualified to perform molecular screening for NRG1 fusions. To initiate pre-screening, a patient must meet one of the following criteria:Histological diagnosis of IMA and documented absence of EGFR/ALK alterations. Note: IMA patients who have not performed the pre-screening test for NRG1 fusion can enter the trial.ORPathological examination does not allow IMA diagnosis but the investigator suspects the IMA based on symptoms, imaging features (e.g. localized consolidation, multiple bilateral nodules or consolidations), non-smoker and documented absence of EGFR/ALK alterations.

The molecular pre-screening Informed Consent Form (ICF) must be signed by NSCLC patients identified for potential study participation before the fresh or archival tumor tissue is submitted for analysis for determination of NRG1 fusion status. Testing can be performed at any time of the natural history of the disease (e.g. at diagnosis, during the first line of therapy, at progression, etc) up to a maximum of one year prior to Cycle 1 Day 1. A fresh tumor sample (formalin-fixed paraffin-embedded; FFPE) or an archival tumor sample not older than 1 year, is required for the assessment of the presence of the NRG1 fusion. The sample should be submitted to a local laboratory qualified for testing by molecular profiling (PCR, next generation sequencing [DNA or RNA] or FISH) of NRG1 fusion status. Patients with a positive local NRG1 fusion result are then eligible to sign the main study ICF if they are willing and able to enter the main study.

Main Informed Consent Form

The main study ICF must be signed by all patients prior to any screening procedures or assessments being conducted. The screening assessments are performed within 4 weeks prior to Cycle 1 Day 1, with the exception of the serum pregnancy test which must be conducted within 7 days of Cycle 1 Day 1. To be considered for screening, a baseline mandatory tumor sample, preferably a block, from fresh or archival tissue is requested. The sponsor indicates the preference for fresh tissue. Archival is acceptable and should have been taken within 2 years from screening other than for NSCLC which must be within 1 year. It should be noted that for NSCLC patients, the baseline biopsy for screening is still required even if a pre-screening biopsy sample is provided for pre-screening local testing of NRG1. Following completion of all required screening assessments and confirmation of all eligibility criteria the patient can begin dosing on Cycle 1 Day 1.

Safety Assessments

Concurrent illnesses are captured at baseline; AEs and concomitant therapies are monitored throughout study participation. Safety assessments include reviewing Eastern Cooperative Oncology Group (ECOG) performance status, physical examination (including height and weight), vital signs and electrocardiograms (ECG). A cardiac function test of the Left Ventricular Ejection Fraction (LVEF) is also be carried out at Screening, end of Cycle 4 (or Cycle 5 Day 1), End of Study Visit, and at any time during the study if clinically indicated. Laboratory evaluations include clinical chemistry, hematology, coagulation tests, urinalysis and pregnancy testing. Note that a cytokine panel analysis was performed up until 1 Aug. 2017.

On all MCLA-128 administration days, the patients must remain at the clinic for at least 60 minutes from the time of the end of infusion (longer where there are PK samples required) for observation and repeat vital signs prior to discharge from the clinic. Further additional safety assessments should be performed as clinically indicated and, if needed, duration of stay in clinic should be increased based on Investigator's judgment.

Immunogenicity Assessment

Serum titers of anti-MCLA-128 antibodies are measured on Day 1 at pre-dose for each of Cycles 1, 2, 3, 4 and then every fourth cycle thereafter (Cycle 8, 12, 16 etc), and at the End of Treatment Visit and the Final Study Visit with a window of −3 days prior to the MCLA-128 administration.

Pharmacokinetics Assessment

Part 1 and Part 2 initial recommended dose schedule: In Cycle 1, blood samples are collected for PK analysis on Day 1 at pre-dose, at end of infusion (EOI), and at 1, 2, 4, 8, 24 hours post EOI, then on Day 4 (or Day 3), Day 8 and Day 15. In Cycles 2-4, only a pre-dose and EOI blood sample is collected.

Part 2 weekly recommended dose schedule: In Cycle 1, blood samples are collected for PK analysis on Day 1 at pre-dose, EOI, 2, 4, 24 hours post EOI, then predose on Days 8 and 15, and predose and EOI on Day 22. In Cycles 2 and 3, a pre-dose and EOI blood sample is collected on Day 15. In Cycle 4 blood samples are collected pre-dose on Day 1, and pre-dose and EOI on Day 15. Every 2 cycles thereafter (Cycles 6, 8, 10 etc) a pre-dose blood sample is collected on Day 15.

Tumor Assessment

Tumor assessment is evaluated according to RECIST version 1.1 per local investigator. Imaging is obtained at Screening and at the end of every 2 cycles of treatment for patients receiving the 3-week cycle regimen and every 6 weeks for patients receiving the 4-week cycle regimen.

Biomarker and Pharmacodynamics Assessments

A range of biomarker and pharmacodynamic tests are performed on archived and/or fresh tumor sample material and/or blood (liquid biopsy), depending on availability of archived or existing tumor tissue, consent for further tumor samples, and consent for specific biomarker testing.

The following candidate biomarkers are assessed in case sufficient sample is available:HER2, HER3, HER2:HER3 dimerization, phosphorylated HER2 (pHER2) and HER3 (pHER3) and heregulin;Circulating plasma tumor DNA (ctDNA) and tumor sample DNA (depending on availability) are used to examine mutations in cancer genes including those associated with HER2 and HER3 signalingPhosphorylated molecules in the MAPK and AKT signaling pathway;Fcgamma receptor polymorphism;Circulating tumor cells for HER2;Heregulin-gene fusions

No germ line DNA assessment is included (except for Fcgamma receptor polymorphism).

At baseline the patient is requested to provide a mandatory tumor sample tissue, preferably a block, which could be from fresh or archival tissue. The sponsor indicates the preference for fresh tissue. Archival is acceptable and should have been taken within 2 years from screening other than for NSCLC which must be within 1 year. In addition the patient is requested optionally to provide a tumor sample/biopsy at the end of Cycle 4 and optionally at the End of Treatment Visit.

Blood samples are also taken at these time points for the purpose of liquid biopsy testing.

Eligibility Criteria:

The study enrolls patients with NSCLC.

General Inclusion Criteria for Part 2

1. Age 18 years or older;2. At least one measurable lesion according to RECIST v1.1;3. Performance status of ECOG 0 or 1;4. Estimated life expectancy of at least 12 weeks;5. Toxicities incurred as a result of previous anti-cancer therapy resolved to ≤Grade 1 (as defined by NCI CTCAE v4.03), except for alopecia, lymphopenia assessed as non-clinically significant, Grade 2 sensory neurotoxicity;6. At least a 4-week interval between the last received radiotherapy and the first scheduled day of dosing with MCLA-128 (with the exception of up to 1×8 Gy for pain palliation);7. Complete recovery from major surgery (stable and <Grade 2 toxicity acceptable);8. Laboratory values at Screening:a. Absolute neutrophil count ≥1.5×109/L without colony stimulating factor support;b. Platelets ≥100×109/L;c. Hemoglobin ≥9 g/dL or ≥2.2 mmol/L (not transfusion dependent);d. Total bilirubin <1.5 times the upper limit of normal (ULN) (unless due to Gilbert's syndrome);e. AST (SGOT)≤2.5×ULN; ALT (SGPT)≤2.5×ULN; ≤5×ULN for patients with advanced solid tumors with liver metastases; patients with confirmed bony metastases are permitted on study with isolated elevations in ALP>5×ULN;f. Serum creatinine ≤1.5×ULN or estimated glomerular filtration rate (GFR) of >50 mL/min based on the Cockroft-Gault formula;g. Coagulation function (INR and aPTT≤1.5 ULN, unless on therapeutic anticoagulants)h. Urine protein ≤2+(as measured by dipstick) or ≤100 mg/24 hours urine;9. Able to provide at baseline a mandatory tumor biopsy sample (FFPE), preferably a block, from fresh (preferred) or archival tissue. Archival tissue must be collected within 2 years before screening, other than for NSCLC which must be within 1 year.10. Negative pregnancy test results available as defined by urine or blood human chorionic gonadotropin (hCG) test during Screening and within 7 days of Cycle 1, Day 1 in women of childbearing potential (defined as women ≤50 years of age or history of amenorrhea for ≤12 months prior to study entry);11. Sexually active male and female patients of childbearing potential must agree to use an effective method of birth control (e.g., barrier methods with spermicides, oral or parenteral contraceptives and/or intrauterine devices) during the entire duration of the study and for 6 months after final administration of MCLA-128. Note that sterility in female patients must be confirmed in the patients' medical records and be defined as any of the following: surgical hysterectomy with bilateral oophorectomy, bilateral tubular ligation, natural menopause with last menses >1 year ago; radiation induced oophorectomy with last menses >1 year ago; chemotherapy induced menopause with 1 year interval since last menses;12. Ability to give written, informed consent prior to any study-specific Screening procedures, with the understanding that the consent may be withdrawn by the patient at any time without prejudice;13. Capable of understanding the mandated and optional protocol requirements, is willing and able to comply with the study protocol procedures and has signed the main informed consent document. For any optional biopsy sampling (tissue and/or blood) and long-term sample storage, additional consent is required;14. Patient with metastatic cancer who has disease progression after having received treatment with all available therapies known to convey clinical benefit.15. Unresectable or metastatic NSCLC meeting one of the following conditions:Biopsy-proven invasive mucinous adenocarcinoma (IMA). Note: IMA patients who have not performed the pre-screening test for NRG1 fusion can enter the trial.ORNSCLC with documented NRG1 fusion determined at in a qualified local laboratory by molecular profiling using methods such as PCR, next generation sequencing [DNA or RNA] or FISH in patients with no known driver mutations or fusions in EGFR/ALK genes.16. Documented disease progression by investigator assessment on at least one line of standard therapy in the locally advanced or metastatic setting
Statistical Analysis:
Part 1 and Part 2

Anti-tumor and clinical benefit variables are summarized descriptively for each group in Part 2. Where appropriate, variables are presented in terms of absolute and relative change from baseline. Categorical data is presented as percentages and frequency tabulations.

Where appropriate, data from those patients who receive what becomes identified as the MTD or the MRD during Part 1, and those receiving the same dose in Part 2, may be combined and summarized, as well as being summarized independently.

The frequency and nature of serious and non-serious AEs is assessed in absolute and relative frequencies and coded according to MedDRA medical dictionary.

Part 1

Data evaluation is descriptive in nature. Patient demographics, disease characteristics and pharmacokinetic and pharmacodynamic variables are summarized at each dose level. The frequency and nature of DLTs are also summarized at each dose level.

Part 2

With N=20 per cohort in Part 2, clinically meaningful observed correlation coefficients of at least 0.38 would be distinguishable from zero with 95% confidence; lesser, non-clinically meaningful observed correlations would not be distinguishable from zero. Hence 20 subjects per cohort in Part 2 is considered sufficient to explore the relationship between the anti-tumor activity of MCLA-128 and disease related biomarkers.

In the event that signs of clinical activity are seen, additional patients up to a total of approximately 40 may be recruited. With 40 patients, true clinical response rates of, for example, 10% to 50% can be estimated with reasonable precision of approximately ±5% to ±8%.

PK parameters are summarized for each cohort in Part 1 and each tumor group in Part 2. Arithmetic and geometric means are provided in addition to medians, range, SD and % CV. AUC is calculated according to the trapezoid rule. Serum concentration profiles against time are plotted for each group.

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