A Phase 2, open-label, multicenter international study will be performed to evaluate the efficacy of MCLA-128-based combinations. Three combination treatments will be evaluated, two in Cohort 1 and one in Cohort 2.

MCLA-128 (zenocutuzumab) is given in combinations in two metastatic breast cancer (MBC) populations, Human Epidermal Growth Factor Receptor (HER) 2-positive/amplified (Cohort 1) and Estrogen Receptor-positive/low HER2 expression (Cohort2).

Two combinations treatments will be evaluated in Cohort 1, the doublet and triplet. Initially zenocutuzumab is given in combination with trastuzumab in the doublet. After the safety of the doublet has been assessed in 4-6 patients, MCLA-128 is given in combination with trastuzumab and vinorelbine in the triplet, in parallel to the efficacy expansion of the doublet.

The doublet and triplet combinations are both evaluated in two steps with an initial safety run-in followed by a cohort efficacy expansion. In total up to 40 patients evaluable for efficacy are included in both the doublet and triplet.

In Cohort 2 zenocutuzumab is administered in combination with the same previous endocrine therapy on which progressive disease is radiologically documented. A total of up to 40 patients evaluable for efficacy are included in the Cohort 2.

Study Design Phase 2, open-label, multicenter international study to evaluate the efficacy of MCLA-128 (zenocutuzumab)-based combinations in 2 metastatic breast cancer populations, Human Epidermal Growth Factor Receptor (HER)2-positive/amplified (Cohort 1) and estrogen receptor-positive/low-HER2 expression (Cohort 2). Three combination treatments were evaluated, 2 in Cohort 1 and 1 in Cohort 2.

Cohort 1: To be eligible, patients had to have HER2-positive/amplified metastatic breast cancer, with confirmed HER2 overexpression by Immunohistochemistry (IHC) with a score of 3+ or of 2+ combined with positive Fluorescence in Situ Hybridization (FISH), have received up to 5 lines of HER2-directed therapy in the metastatic setting, and have progressed on the most recent line per Response Evaluation Criteria In Solid Tumors Guidelines (RECIST) v1.1, and have been previously treated with trastuzumab, pertuzumab and an HER2 Antibody-Drug Conjugates (ADC) in any sequence and any setting.

Initially zenocutuzumab was administered with trastuzumab (doublet combination). Safety was reviewed by an Independent Data Monitoring Committee (IDMC). If the safety of the doublet was approved, the triplet combination of zenocutuzumab plus trastuzumab and vinorelbine was to be evaluated in parallel with the doublet combination.

The doublet and triplet Cohort 1 combinations were each evaluated in 2 steps with an initial safety run-in period in 4 to 6 patients who were reviewed by the IDMC before deciding to expand the cohort. The triplet combination go/no-go decision was made by the IDMC after evaluation of the doublet safety run-in patients (based on Adverse Avents [AEs], Serious Adverse Events [SAEs], relationship to study drug, and other clinically relevant parameters [eg, laboratory parameters], available pharmacokinetics, immunogenicity, and cytokine data). If the triplet combination was considered safe, the expansion of the doublet and triplet combinations was performed in parallel. Patients were included in the triplet or doublet in a 3:1 ratio, taking into account previous exposure to vinorelbine.

After the safety run-in, if Cohort 1 doublet and Cohort 1 triplet combination therapies were considered tolerable by the IDMC, they were each to be expanded to a total of up to 40 patients evaluable for efficacy. If the doublet combination regimen was not well tolerated, Cohort 1 was to be closed. If the triplet combination was not well tolerated but the doublet was acceptable, the doublet expansion was to be continued.

Cohort 2: To be eligible, patients had to have estrogen receptor-positive and low-HER2 expression metastatic breast cancer (IHC 1+, or IHC 2+ combined with negative FISH), and radiologic or photographic evidence of disease progression on the last line of prior endocrine therapy (administered for ≥12 weeks) that included an aromatase inhibitor (AI) or fulvestrant. Patients who had received up to 3 prior endocrine therapies in the metastatic setting and had progressed on a Cyclin-Dependent Kinase (CDK) inhibitor (in any line) were eligible.

Zenocutuzumab was administered in combination with the same previous endocrine therapy on which progressive disease was radiologically/photographically documented. Up to 40 patients evaluable for efficacy were included.

Inclusion Criteria

1. Signed informed consent before initiation of any study procedures.

2. Women with histologically or cytologically confirmed breast cancer with evidence of metastatic or locally advanced disease not amenable to any local therapy with curative intent:

   2.1 Cohort 1 (zenocutuzumab + trastuzumab ± vinorelbine)

   1. Documented Human Epidermal Growth Factor Receptor (HER)2 overexpression/amplification, defined as Immunohistochemistry (IHC) 3+ positive, or IHC 2+ combined with positive Fluorescence In Situ Hybridization (FISH), based on local analysis on the most recent tumor biopsy (preferably metastatic, otherwise primary), either fresh or archival collected within 24 months before screening.
   2. Documented disease progression (by investigator assessment) on up to a maximum of 5 lines of HER2- directed therapy administered in the metastatic setting and progression on the most recent line. Trastuzumab, pertuzumab and an HER2 Antibody drug conjugates (ADC) (eg. Trastuzumab emtansine (T-DM1)) must have been previously administered in any sequence and in any setting.

   2.2 Cohort 2 (zenocutuzumab + endocrine therapy)

   1. Documented hormone receptor positive status (estrogen receptor positive and/or progesterone receptor positive), defined as ≥ 1% positive stained cells by local standards, based on local analysis on the most recent tumor biopsy.
   2. Documented low-level HER2 expression, defined as IHC HER2 1+, or IHC HER2 2+ combined with negative FISH, based on local analysis on a fresh tumor biopsy or an archival biopsy collected within 24 months before screening (preferably metastatic, otherwise primary).
   3. No more than 3 lines of prior endocrine therapy (aromatase inhibitor or fulvestrant) for metastatic disease, with radiologic or photographic evidence of disease progression on the last line, after at least 12 weeks of therapy.
   4. Progression on a cyclin-dependent kinase inhibitor.
   5. No more than 2 previous chemotherapy regimens for advanced/metastatic disease. Note: Pre/peri-menopausal women could be enrolled if amenable to be treated with the Luteinizing Hormone-Releasing Hormone (LHRH) agonist goserelin. Such patients must have commenced treatment with goserelin or an alternative LHRH agonist at least 4 weeks prior to study entry, and patients who received an alternative LHRH agonist prior to study entry must switch to goserelin for the duration of the trial.

3. At least one lesion with measurable disease as defined by Response Evaluation Criteria In Solid Tumors Guidelines (RECIST) v1.1. For Cohort 2, patients with bone-only disease were eligible, even in the absence of measurable disease. Patients with bone-only disease must have lytic or mixed lesions (lytic + sclerotic), and imaging documenting progression on the last line of hormone therapy must be available for central review.

4. Age ≥ 18 years at signature of informed consent.

5. Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.

6. Life expectancy of ≥ 12 weeks, as per investigator.

7. Left ventricular ejection fraction (LVEF) ≥50% by echocardiogram (ECHO) or multiple gated acquisition scan (MUGA).

8. Adequate organ function:

   1. Absolute neutrophil count (ANC) ≥ 1.5 X 109/L.
   2. Hemoglobin ≥ 9 g/dL.
   3. Platelets ≥ 100 x 109/L.
   4. Serum calcium within normal ranges (or corrected with supplements).
   5. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) ≤2.5 x upper limit of normal (ULN) and total bilirubin ≤1.5 x ULN (in cases of liver involvement, ALT/AST ≤5 x ULN and total bilirubin within normal ranges was allowed).
   6. Serum creatinine ≤1.5 x ULN or creatinine clearance ≥ 60 mL/min calculated according to the Cockcroft and Gault formula or Modification of Diet in Renal Disease (MDRD) formula for patients aged >65 years (Protocol Appendix 19.2).
   7. Serum albumin >3.0 g/dL.

Exclusion Criteria

1. Central nervous system metastases that are untreated or symptomatic, or require radiation, surgery, or continued steroid therapy to control symptoms within 14 days of study entry.

2. Known leptomeningeal involvement.

3. Advanced/metastatic, symptomatic, visceral spread, with a risk of life-threatening complications in the short-term (including patients with massive uncontrolled effusions [pleural, pericardial, peritoneal], pulmonary lymphangitis, and over 50% liver involvement).

4. Participation in another interventional clinical trial or treatment with any investigational drug within 4 weeks prior to study entry.

5. Any systemic anticancer therapy within 3 weeks of the first dose of study treatment. For cytotoxic agents that have major delayed toxicity, eg, mitomycin C and nitrosoureas, or anticancer immunotherapies, a washout period of 6 weeks was required. For patients in Cohort 2, this did not apply to the most recently received hormone therapy.

6. Major surgery or radiotherapy within 3 weeks of the first dose of study treatment. Patients who received prior radiotherapy to ≥25% of bone marrow were not eligible, irrespective of when it was received.

7. Persistent grade >1 clinically-significant toxicities related to prior antineoplastic therapies (except for alopecia); stable sensory neuropathy ≤ Grade 1 National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) v4.03 was allowed.

8. History of hypersensitivity reaction or any toxicity attributed to trastuzumab, murine proteins, or any of the excipients that warranted permanent cessation of these agents (applicable for Cohort 1 only).

9. Previous exposure to vinorelbine (applicable for Cohort 1 triplet combination only).

10. Exposure to the following cumulative anthracycline doses:

    1. Doxorubicin or liposomal doxorubicin >360 mg/m².
    2. Epirubicin >720 mg/m².
    3. Mitoxantrone >120 mg/m² and idarubicin >90 mg/m².
    4. Other anthracycline at a dose equivalent to >360 mg/m² doxorubicin
    5. For patients having received > 1 anthracycline, the cumulative dose must not exceed the equivalent of 360 mg/m² doxorubicin.

11. Chronic use of high-dose oral corticosteroid therapy (>10 mg of prednisone equivalent per day).

12. Uncontrolled hypertension (systolic >150 mmHg and/or diastolic > 00 mmHg) or unstable angina.

13. History of congestive heart failure of Class II-IV New York Heart Association (NYHA) criteria, or serious cardiac arrhythmia requiring treatment (except atrial fibrillation, paroxysmal supraventricular tachycardia).

14. History of myocardial infarction within 6 months of study entry.

15. History of prior or concomitant malignancies (other than excised non-melanoma skin cancer or cured in situ cervical carcinoma) within 3 years of study entry.

16. Current dyspnea at rest of any origin, or other diseases requiring continuous oxygen therapy.

17. Current serious illness or medical conditions including, but not limited to uncontrolled active infection, clinically significant pulmonary, metabolic, or psychiatric disorders.

18. Known human immunodeficiency virus (HIV), hepatitis B virus (HBV), or hepatitis C virus (HCV) infection.

19. Pregnant or lactating women; women of childbearing potential must use effective contraception methods (patient and/or partner, eg, surgical sterilization, a reliable barrier method) prior to study entry, for the duration of study participation, and for 7 months after the last dose of zenocutuzumab/trastuzumab. See Protocol Section 8.10.

20. Patients with only non-measurable lesions other than bone metastasis (eg, pleural effusion, ascites, or other visceral locations).

21. Patients with bone-only disease with blastic-only metastasis.