INHIBITORS OF CHI3L1 AND THEIR USES

Abstract

The present invention relates to a suppressor or inhibitor of the expression and/or the activity of Chitinase 3-like 1 (CHI3L1) for use in the prevention and/or treatment of tumors, wherein said tumors are resistant to antibody-dependent cell-mediated cytotoxicity (ADCC) dependent therapies and/or for use in reducing the risk of developing resistance to antibody-dependent cell-mediated cytotoxicity (ADCC) dependent therapies.

Claims

1: A method for suppressing or inhibiting the expression and/or the activity of a Chitinase 3-like 1 (CHI3L1) for: preventing or treating tumors, wherein said tumors are resistant to antibody-dependent cell-mediated cytotoxicity (ADCC) dependent therapies, and/or for reducing the risk of developing resistance to antibody-dependent cell-mediated cytotoxicity (ADCC) dependent therapies, the method comprising administering to an individual in need thereof a inhibitor or suppressor of CHI3L1, wherein the suppressor or inhibitor comprises at least one molecule selected from the group consisting of: a) an antibody or a fragment thereof; b) a polypeptide; c) a small molecule; d) a polynucleotide coding for said antibody or polypeptide or a functional derivative thereof; e) an inhibitory polynucleotide, f) a vector comprising or expressing the polynucleotide as defined in d) or e); g) a CRISPR/Cas9 component, optionally a sgRNA; and h) a host cell genetically engineered expressing said polypeptide or antibody or comprising the polynucleotide as defined in d) or e) or at least one component of g).

2: The method of claim 1, wherein the CHI3L1 is extracellular, or tumor-derived and/or wherein the tumor expresses CHI3L1 at high levels.

3: The method of claim 1, wherein the tumor is a tumor which is resistant to anti-HER2, or to anti-CD20, or to anti-GD20, or to anti-CCR4, or to anti-EGFR, or to anti-CD19 or to anti-MUC1 or to anti-GA201 antibody therapies, and optionally the tumor is resistant to Trastuzumab, Pertuzumab, Rituximab, Dinituximab, Obinutuzumab, Mogamulizumab, Cetuximab and/or Panitumumab.

4: The method of claim 1, wherein the tumor comprises HER2 positive cancer cells, and optionally the tumor is a breast tumor or a gastric cancer, and is resistant to anti-HER2 antibody therapies, and optionally the tumor is resistant to Trastuzumab and/or to Pertuzumab.

5: The method of claim 1, wherein the inhibitory polynucleotide comprises an antisense construct, an antisense oligonucleotide, an RNA interference construct or an siRNA.

6: The method of claim 1, wherein said suppressor or inhibitor comprises an anti-CHI3L1 antibody, optionally a monoclonal antibody, and optionally the antibody is derived from the clone mAY, or said suppressor or inhibitor comprises an inhibitor of a CHI3L1 receptor, or the soluble form of a CHI3L1 receptor, and optionally the inhibitor or suppressor comprises an interleukin 13 receptor alpha 2 (IL13ra2), or a Receptor for Advance Glycation End product (RAGE).

7: The method of claim 6, wherein the tumor is resistant to Trastuzumab.

8: The method of claim 1, wherein the suppressor or inhibitor is used in combination with at least one therapeutic antibody which is capable of antibody-dependent cell-mediated cytotoxicity (ADCC), optionally an anti-HER2, an anti-CD20, an anti-GD20, an anti-CCR4, an anti-EGFR, an anti-CD19 or an anti-MUC1 or an anti-GA201 antibody, optionally with Trastuzumab, Pertuzumab, Rituximab, Dinituximab, Obinutuzumab, Mogamulizumab, Panitumumab and/or Cetuximab.

9: A pharmaceutical composition comprising: i. at least one suppressor or inhibitor of expression and/or activity of Chitinase 3-like 1 (CHI3L1), ii. at least one therapeutic antibody which is capable of antibody-dependent cell-mediated cytotoxicity (ADCC) and iii. at least one pharmaceutically acceptable excipient, diluent or carrier, for use in the prevention and/or treatment of tumors, wherein said tumors are resistant to antibody-dependent cell-mediated cytotoxicity (ADCC) dependent therapies and/or for use in reducing the risk of developing resistance to antibody-dependent cell-mediated cytotoxicity (ADCC) dependent therapies, and optionally the at least one therapeutic antibody comprises at least one anti-HER2 antibody, an anti-CD20 antibody, an anti-GD20 antibody, an anti-CCR4 antibody, an anti-EGFR antibody, an anti-CD19 antibody or an anti-MUC1 antibody or an anti-GA201 antibody, and optionally the at least one therapeutic antibody is selected from the group consisting of: Trastuzumab, Pertuzumab, Rituximab, Dinituximab, Obinutuzumab, Mogamulizumab, Panitumumab and/or Cetuximab, more preferably Trastuzumab and/or Pertuzumab, wherein the suppressor or inhibitor of expression and/or activity of CHI3L1 comprises at least one molecule selected from the group consisting of: a) an antibody or a fragment thereof; b) a polypeptide; c) a small molecule; d) a polynucleotide coding for said antibody or polypeptide or a functional derivative thereof; e) an inhibitory polynucleotide, f) a vector comprising or expressing the polynucleotide as defined in d) or e); g) a CRISPR/Cas9 component, optionally a sgRNA; and h) a host cell genetically engineered expressing said polypeptide or antibody or comprising the polynucleotide as defined in d) or e) or at least one component of g).

10: The pharmaceutical composition of claim 9, further comprising a therapeutic agent, wherein said therapeutic agent is for the treatment and/or prevention of a tumor.

11: The pharmaceutical composition of claim 9, wherein the tumor is a tumor which is resistant to anti-HER2, or to anti-CD20, or to anti-GD20, or to anti-CCR4, or to anti-EGFR, or to anti-CD19 or to anti-MUC1 or to anti-GA201 antibody therapies, and optionally the tumor is resistant to Trastuzumab, Pertuzumab, Rituximab, Dinituximab, Obinutuzumab, Mogamulizumab, Cetuximab and/or Panitumumab.

12: The pharmaceutical composition of claim 9, wherein the tumor comprises HER2 positive cancer cells, and optionally the tumor is a breast tumor or a gastric cancer, and is resistant to anti-HER2 antibody therapies, and optionally the tumor is resistant to Trastuzumab and/or to Pertuzumab.

13: An in vitro method for identifying a patient with a tumor which is resistant to antibody-dependent cell-mediated cytotoxicity (ADCC) dependent therapies, comprising the steps of: a) detecting CHI3L1 in an isolated biological sample obtained from the patient and b) comparing with respect to a proper control, wherein an amount of CHI3L1 in the isolated biological sample obtained from the subject higher than the control amount indicates that tumor is resistant to antibody-dependent cell-mediated cytotoxicity (ADCC) dependent therapies.

14: An in vitro method for prognosing and/or diagnosing and/or assessing the risk of developing and/or for monitoring the progression and/or for monitoring the efficacy of a therapeutic treatment and/or for the screening of a therapeutic treatment of a tumour resistant to antibody-dependent cell-mediated cytotoxicity (ADCC) dependent therapies in a subject comprising the steps of: a) detecting a CHI3L1 in an isolated biological sample obtained from the subject and b) comparing the results of (a) with respect to a proper control.

15: The in vitro method according to claim 13 wherein the tumor comprises HER2 positive cancer cells, and optionally the tumor is a breast tumor or a gastric cancer, and is resistant to anti-HER2 antibody therapies, and optionally to tumor it is resistant to Trastuzumab and/or to Pertuzumab.

16: The in vitro method of claim 14, wherein the tumor comprises HER2 positive cancer cells, and optionally the tumor is a breast tumor or a gastric cancer, and is resistant to anti-HER2 antibody therapies, and optionally to tumor it is resistant to Trastuzumab and/or to Pertuzumab.

Description

[0219] The invention will be illustrated by means of non-limiting examples in reference to the following figures.

[0220] FIG. 1: ADCC of NK cells from healthy donors is negatively modulated by sera of Trastuzumab resistant patients (PD) but not complete remission patients (RC)

[0221] FIG. 2: CHI3L1 levels correlate with disease progression and unfavorable prognosis In breast cancer. (a) levels of CHI3L1 in the sera of HER2 breast cancer PO patients (N=10), CR patients (N=10) and healthy controls (N=9) during treatment (Left pane) or at study endpoint (right panel), (b) Overall survival of HER-2 positive breast cancer patients according to CHI3L1 expression in primary tumor (n=95, Cutoff=Median), source: Kmplotter (breast cancer), (c) overall survival of breast cancer patients from all subtypes according to CHI3L1 gene expression in primary tumor (n=508, Cutoff=Median), source: E-GEOD-25066. (d) and (e) Relapse free survival of breast cancer patients according to CHI3L1 gene expression in primary tumor (Cutoff=most significant), source: E-GEOD-42568 and GSE29271 respectively.

[0222] FIG. 3: CHI3L1 inhibits ADCC and Natural cytotoxicity of NK cells and CD8 T cells by impairing lytic granules polarization to the immune synapse. (a) Dose dependent inhibition of NK cell ADCC by rhCHI3L1 against SKBR3 Her2 overexpressing target cells, (b) Effect of 50 ng/ml rhCHI3L1 on NK cell ADCC from different healthy donors (N=7). (c) Effect of 50 ng/ml rhCHI3L1 on natural cytotoxicity of NK cells against K562 MHC-I missing targets (N«5 healthy donors), (d) Effect of rhCHI3L1 on CD8 T cell cytotoxicity against MEC-1 cells loaded with super-antigen (SEB). (e) Effect of 50 ng/ml rhCHI3L1 on murine NK cells ADCC (N=6 mice), (f) Quantification of confocal microscopy images showing frequencies of correctly polarized lytic granules in NK cells conjugated in ADCC with SKBR3 target cells (N=number of NK conjugates counted from 4 healthy donors). All plots are representative of at least two independent evaluations. Values in all graphs represent the means f SD (95% Cl, *p<0.05, **p<0.01, ***p<0.001, NS, not significant).

[0223] FIG. 4: NK cell ADCC and trastuzumab efficacy is restored by blocking CHI3L1 activity in vitro.

[0224] FIG. 5: CHI3L1 binds RAGE and blocks its downstream JNK signaling.

[0225] (a) Dot plots for RAGE expression on NK cells (left) and MFI of RAGE expression on NK cells treated with rhCHI3L1. (b) MFI of lytic granules staining in NKs treated with CHI3L1 (50 ng/ml) or anti-RAGE Blocking antibody (10 ug/ml) for 1 h. (c) effect of RAGE blockade on NK cells cytotoxicity (d) Basal P-JNK levels in NK cells treated with CHI3L1 (e) P-JNK levels in NK cells after 10 min of stimulation by RAGE ligand S100A8/A9 (10 ug/ml) alone or with rhCHI3L1 (100 ng/ml) and (f) P-JNK levels in time during ADCC of NK cells incubated with SKBR3 targets at (5:1) E:T ratio. All plots are representative of at least two

[0226] independent evaluations. Values in all graphs represent the means±SD (95% Cl, *p<0.05, **p<0.01, ***p<0.001, NS, not significant)

[0227] FIG. 6: CHI3L1 injections increase tumor take and enhance the growth of NK-sensitive tumors.

[0228] RMA-S lymphoma cells at limiting number (4×10.sup.5) were injected s.c in the flank of female C57BL/6 mice at D0. Mice were randomized in three groups to receive PBS, rmCHI3L1 (0.5 ug/mouse i.p every other day starting D0), or anti-NK1.1 antibody for NK cell depletion (150 ug/mouse i.p on D0, D2, D4). Tumor take and tumor growth were scored every two days. After sacrifice, tumors were weighted and imaged.

[0229] FIG. 7. CHI3L1 overexpression abrogates the efficacy of Trastuzumab ADCC to control JIMT-1 tumors

[0230] 3×10.sup.6 JIMT-1 cells overexpressing CHI3L1 (JIMT-1CHI311) or 4×10.sup.6 cells transfected with empty plasmid (JIMT-1 Mock) were injected subcutaneously in the flank of BALB/c Nude mice. The injection of a higher number of JIMT-1 Mock cells was done to compensate for the faster tumor growth of CHI3L1 overexpressing ones. I.p Treatment with 10 mg/kg Trastuzumab or hIgG isotype control was started when tumors reached 50 mm.sup.3 and was given once every week. Tumor take and tumor growth were scored every two days. After sacrifice, tumors were weighted and imaged.

[0231] FIG. 8. Neutralizing CHI3L1 boosts the efficacy of Trastuzumab by unleashing NK cells.

[0232] HCC1569 cells (2×10.sup.6) were injected in the flank of NSG mice. Once tumors reached 50 mm.sup.3 mice were assigned into the different treatment groups. 8×10.sup.6 expanded human NK cells were transferred via the tail vein to mice in some groups (D0). Anti-CHI3L1 treatment was started on D-1 (200 ug/mouse every two days) and Trastuzumab treatment was started on D1 (200 ug/mouse once a week) of NK cell transfer. Experiment was stopped when Isotype control treated mice developed necrotic tumors at which time the Trastuzumab+NK cell+anti-CHI3L1 mice had two tiny Tumors remaining. Mice were then sacrificed and tumors were imaged.

DETAILED DESCRIPTION OF THE INVENTION

Examples

Example 1

Materials and Methods

Patients

[0233] Women with histological diagnosis of locally advanced invasive or metastatic BC were considered eligible for the study if classified as HER-2 positive, i.e. score 3+ (by immuno-histochemical analysis: IHC) or IHC score 2+ and FISH (fluorescence in situ hybridization) amplified. Thirty BC patients were enrolled in the study that underwent a first line chemotherapy in combination with trastuzumab. Response to trastuzumab was evaluated on the basis of clinical, pathological and radiologic examination of the tumor before and after treatment. The revised RECIST criteria (version 1.1) were used to evaluate the treatment response which was classified as complete response (CR) and disease progression (PD). Blood samples were collected at basal (before Trastuzumab therapy) and subsequently until patients reached PD or RC state (12 months).

[0234] This study was approved by the Ethics Committee of the European institute of oncology hospital, Milan, Italy and written informed consent was obtained from each patient.

Cytotoxicity Assay

[0235] NK cell and CD8 T cell-mediated cytotoxicity was determined using the DELFIA® EuTDA Cytotoxicity Reagents (PerkinElmer Life Sciences, Waltham, Mass., USA), according to manufacturer's instructions. Briefly, target cells (1×10.sup.6 cells/ml) were incubated with freshly prepared 10 μM BATDA (a fluorescence enhancing ligand) in 2 ml of culture medium for 30 min at 37° C., and washed. Next, 100 μl of BATDA-labeled target cells (5,000 cells) were transferred into a V-bottom sterile plate. In ADCC assays SKBR3 cells (commercial from ATCC, HTB30) were treated with 10 ug/ml Trastuzumab (Roche) for 20 min at 37° C. and in primary CD8 T cells [from healthy donors, after informed consent] cytotoxicity MEC1 targets (IEO cell line bank) were loaded with 2 ug/ml SEB sAg (SIGMA) for 30 min at 37° C. Effector cells were treated as described in each experiment and then co-cultured wit target cells for 1 h 30 min in case of ADCC and 2 hours in case of natural NK cytotoxicity or CD8 T cells cytotoxicity at effector/target ratio of 10:1. After incubation, 20 μl of supernatant from each well was transferred to the wells of flat-bottom 96 well plates. 180 μl of europium (Eu) solution was then added to form highly fluorescent and stable chelates (EuTDA), and the fluorescences of these chelates were measured by time-resolved fluorometry (Victor3, PerkinElmer). The percent of specific release was calculated using (experimental release−spontaneous release)/(maximum release−spontaneous release)×100(%). In blocking experiments, anti-CHI3L1 (clone MaY, MABC196, Sigma-Aldrich) or mIgG1 isotype (EMD millipore) or rhsILl3ra2 (abcam) was pre-incubated with the treatment agent (patient Serum or rhCHI3L1) for 1 h before adding the mixture to suspensions of NK cells and incubating for 1 h prior to co-cultured with target cells. All experiments were performed in triplicate.

Flow Cytometry

[0236] Single-cell suspensions, of blood or from cell culture, were obtained and incubated with Fc receptor-blocking antibody before being stained on ice. Dead cells were excluded by live/dead staining using fixable live/dead stain dyes (Thermofisher scientific). Primary NK cells [from healthy donors, after informed consent] were gated as singlets, CD14neg, FSC−. SSC appropriate for lymphocytes, CD3neg, CD16/CD56 poscells. Intracellular staining was performed using a fixation and permeabilization buffer (BD Bioscience). For phosphoflow experiments NK cells were fixed with 4% PFA for 12 min at 37° C. immediately after treatment time and permeabilized with Ice cold PermIII buffer (BDbioscience) for 30 min on ice. Stained cells were analyzed on FACSCanto II cytometer (BD Biosciences). Cell doublets were excluded by comparison of side-scatter width to forward-scatter area. Flow cytometry data were analyzed with FlowJo v10 software (Tree Star).

Results

[0237] To investigate the involvement of NK cell mediated ADCC in trastuzumab resistance, inventors collected sera from metastatic breast cancer patients treated with trastuzumab in adjuvant therapy. NK cells isolated from healthy donors were incubated with the patients' sera and tested for cytotoxic activity. Inventors found that response to trastuzumab can be predicted based on the capacity of serum from treated patients to influence the activity of healthy NK cells where sera from non-responders (N=8) reduced NK cell activity, while sera from responders had no effect on NK cell activity (N=11) (FIG. 1).

[0238] To identify the molecules involved in the observed phenomenon, inventors carried out a comparative proteomic analysis of the sera of responder versus non-responder patients and identified some molecular candidates that may explain both innate and acquired resistance to trastuzumab. Among these, Chitinase 3-like 1 protein (CHI3L1) was found high at baseline in sera of non-responders or increased during therapy in the sera of patients with acquired resistance (FIG. 2a). In line with these findings, high CHI3L1 expression in primary tumors correlated with worse overall survival (OS) and progression free survival (PFS) in different breast cancer public cohorts (FIG. 2b-e).

[0239] To test if CHI3L1 is directly responsible for the decreased cytotoxic activity, we pretreated NK cells with rhCHI3L1 before incubating them with target cells. Inventors found that addition of CHI3L1 impairs both antibody-dependent and natural cytotoxicity of human and murine NK cells in a dose dependent manner (FIG. 3a-c,e). Interestingly, inventors observed a similar inhibition of CD8 T cell cytotoxicity against MEC-1 targets loaded with SEB super-antigen (FIG. 3d). Confocal imaging of the immune synapse between NK cells and target cells revealed an impaired polarization of lytic granules towards the immune synapse in CHI311 treated cells (FIG. 3e)

[0240] To test the possibility of restoring trastuzumab dependent cytotoxicity of NK cells, inventors co-incubated with a CHI3L1 neutralizing antibody (mAY) or soluble rhIL13ra2. Both methods abrogated the inhibitory effect of CHI3L1 (FIG. 4).

[0241] To elucidate the molecular mechanism by which CHI3L1 is acting on NK cells. Inventors first analyzed the expression of receptor of advanced glycation end products (RAGE). Consistent with a previously described activator function of RAGE in murine NK cells inventors found this receptor to be expressed at high levels on CD56+ NK cells. Further, the expression of RAGE was downmodulated after incubation with CHI3L1 suggesting the internalization of RAGE upon CHI3L1 binding (FIG. 5a).

[0242] Inventors next tried to validate the role of RAGE in NK cell cytotoxicity. RAGE blocking antibody, similarly to CHI3L1, inhibited NK cell ADCC and induced a decrease of lytic granules (FIG. 5b,c). This suggested to us that CHI3L1 played an inhibitory rather than an activatory function through RAGE binding. To address this issue, inventors evaluated the effect of CHI3L1 ligation on the signaling pathway downstream of RAGE. Inventors focused on pathways that have been shown to be important for NK cell cytotoxicity, including granules biology and polarization, namely ERK1/2 and JNK. While CHI3L1 treatment did not impact on ERK1/2 phosphorylation levels, basal P-JNK levels were decreased in a dose dependent manner in CHI3L1 treated NK cells (FIG. 4d). Further, CHI3L1 opposed the induction of JNK phosphorylation by the RAGE agonist S100A8/A9, which has been described to activate murine NK cells through RAGE (FIG. 4e). Inventors found that activation of JNK occurred in a time dependent manner during NK cell ADCC and pretreatment with CHI3L1 or anti-RAGE resulted in a significantly lower P-JNK levels during ADCC suggesting suboptimal JNK phosphorylation in CHI3L1 treated cells (FIG. 5f).

Example 2

Materials and Methods

Tumor Models

[0243] Mice were purchased from Charles River Laboratories and were cared for and used under specific pathogen-free conditions according to the guidelines established in the Principles of Laboratory Animal Care (directive 86/609/EEC). RMA-S cells were a kind gift from Dr. Sebastian Kobold (LMU, Munich). JIMT-1 and HCC1569 cell lines were purchased from ATCC. Tumor cells at 70% confluence were washed twice and resuspended in cold PBS and kept on ice during the injection procedure. Tumor measurements and endpoints were registered by an observer blinded to the treatment groups.

Results

[0244] RMA-S tumors are vulnerable to NK cell lysis. Injecting mice with CHI3L1 leads to a drastic increase in tumor establishment and tumor growth similar to that of mice where NK cells have been depleted (anti-NK1.1) (FIG. 6). This suggests that CHI3L1 is shutting down NK cell mediated immune surveillance of tumor growth.

[0245] JIMT-1 tumors are resistant to Trastuzumab in vitro. However, a partial tumor control can be achieved in vivo thanks to NK cell ADCC. Therefore, a dysfunction of NK cell ADCC in this model will directly reflect negatively on the ability of Trastuzumab to control tumor growth. Indeed, JIMT-1 tumors that do not express CHI3L1 (Mock) were controlled by Trastuzumab injections. On the other hand, CHI3L1 overexpression abrogated tumor control where tumors were rendered completely insensitive to Trastuzumab Injections (FIG. 7). HCC1569 HER2+ BC tumor cells endogenously express high levels of CHI3L1. These cells were derived from a Trastuzumab resistant patient although they still respond partially to Trastuzumab mediated HER2 inhibition in vitro. To closely mimic the human therapeutic setting, we injected these cells in NSG mice and transferred human NK cells to these mice, followed by treatment with Trastuzumab alone, or in combination with anti-human CHI3L1 neutralizing antibody (clone MaY). We observed a partial tumor control by Trastuzumab alone. Importantly NK cell transfer did not have any additional benefit suggesting that NK cells are inhibited in these mice. Only when mice were treated with a CHI3L1 neutralizing antibody, NK cell transfer in combination with Trastuzumab led to complete regression of tumors (FIG. 8). These results demonstrate the therapeutic potential of targeting CHI3L1 to restore NK cell activity and boost Trastuzumab (ADCC) Efficacy in a robust pre-clinical model closely mimicking the human disease.

TABLE-US-00004 TABLE 1 Approved therapeutic antibodies where ADCC is a determinant of treatment efficacy: Antibody Target Modification Status Rituximab CD20 FDA approved for non- Hodgkin's lymphoma(Cartron et al., 2002) Dinituximab GD2 FDA approved for high- risk neuroblastoma, combined with IL2 and GM-CSF(Yu et al., 2010) Obinutuzumab CD20 Reduced Completed Clinical fucosylation Trials. FDA Approved for CLL in 2013(Goede et al., 2014) Mogamulizumab CCR4 Afucosylated Completed Clinical Trials. Approved in Japan for T-Cell Lymphoma. FDA Approved for non- Hodgkins lymphoma in 2018(‘Correction: Mogamulizumab versus vorinostat in previously treated cutaneous T-cell lymphoma (MAVORIC): an international, open- label, randomised, controlled phase 3 trial (The Lancet Oncology (2018) 19(9) (1192- 1204), (S1470204518303796) (10.1016/S1470-204’, 2018) Cetuximab EGFR FDA approved for metastatic colorectal cancer and head and neck cancer(Tol et al., 2009) Trastuzumab HER2 FDA approved for HER2+ breast cancer and HER2+ metastatic gastric adenocarcinoma(Junttila et al., 2010)

REFERENCES

[0246] 1. Cartron, G. et al. Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor FcγrIIIa gene. Blood 99, 754-758 (2002). [0247] 2. Yu, A. L. et al. Anti-GD2 Antibody with GM-CSF, Interleukin-2, and Isotretinoin for Neuroblastoma. N. Engl. J. Med. 363, 1324-1334 (2010). [0248] 3. Goede, V. et al. Obinutuzumab plus Chlorambucil in Patients with CLL and Coexisting Conditions. N. Engl. J. Med. 370, 1101-1110 (2014). [0249] 4. Correction: Mogamulizumab versus vorinostat in previously treated cutaneous T-cell lymphoma (MAVORIC): an international, open-label, randomised, controlled phase 3 trial (The Lancet Oncology (2018) 19(9) (1192-1204), (S1470204518303796) (10.1016/S1470-204. The Lancet Oncology 19, e581 (2018). [0250] 5. Tol, J. et al. Chemotherapy, bevacizumab, and cetuximab in metastatic colorectal cancer. [Erratum appears in N Engl J Med. 2010 Dec. 23; 363(26):2573]. N. Engl. J. Med. 360, 563-572 (2009). [0251] 6. Junttila, T. T. et al. Superior in vivo efficacy of afucosylated trastuzumab in the treatment of HER2-amplified breast cancer. Cancer Res. 70, 4481-4489 (2010). [0252] 7. David Zahavi, Dalal AlDeghaither, Allison O'Connell, Louis M Weiner, Enhancing antibody-dependent cell-mediated cytotoxicity: a strategy for improving antibody-based immunotherapy, Antibody Therapeutics, Volume 1, Issue 1, June 2018, Pages 7-12,