Formulation and clinical translation of [¹⁷⁷Lu]Lu-trastuzumab for radioimmunotheranostics of metastatic breast cancer

Abstract

Trastuzumab (Herceptin®) is an approved immunotherapeutic agent used for the treatment of metastatic breast cancer over-expressing HER2 antigen receptors. The aim of the present work is to standardize the formulation protocol of [¹⁷⁷Lu]Lu-trastuzumab addressing various reaction parameters, evaluating the efficacy of the radiolabeled product by in vitro investigations, scaling-up the preparation for administration in patients and performing preliminary clinical studies in patients suffering from metastatic breast cancer. Trastuzumab was conjugated with a suitable bi-functional chelating agent namely, p-NCS-benzyl-DOTA. On average 6.15 ± 0.92 p-NCS-benzyl-DOTA molecules were observed to be attached to each trastuzumab moiety. [¹⁷⁷Lu]Lu-trastuzumab could be prepared with >95% radiochemical purity (% RCP) employing the optimized radiolabeling procedure. In vitro studies revealed the affinity of [¹⁷⁷Lu]Lu-trastuzumab towards HER2 +ve cancer cell lines as well as against HER2 protein (K_d = 13.61 nM and 11.36 nM, respectively). The value for percentage immunoreactive fraction (% IRF) for [¹⁷⁷Lu]Lu-trastuzumab was observed to be 76.92 ± 2.80. Bio-distribution studies in Swiss mice revealed non-specific uptake in the blood, liver, lungs and heart followed by gradual clearance of activity predominantly through the hepatobiliary route. Preliminary clinical studies carried out in 8 cancer patients with immunohistochemically proven HER2 positive metastatic breast cancer revealed preferential localization of [¹⁷⁷Lu]Lu-trastuzumab in breast cancer lesions, which was in concordance with [¹⁸F]FDG-PET scans recorded earlier in the same patient indicating the potential of the agent towards radioimmunotheranostic applications.

Clinical translation of ¹⁷⁷Lu[Lu]-trastuzumab from the laboratory to the clinic for radioimmunotherapy of breast cancer over-expressing HER2 receptors.

Introduction

Immunotherapy is a type of cancer treatment which artificially stimulates the immune system of the human body thereby helping the patient to fight against the disease and is given either in an adjuvant or in a neo-adjuvant manner.^1,2 Radioimmunotherapy (RIT) is a special class of immunotherapy which exploits the affinity of immune proteins towards the tumor-associated specific antigens or antigen receptors for delivering a therapeutic radionuclide to the target of interest in order to provide a lethal dose of cytotoxic radiation to the cancerous lesions.^3–7 RIT finds wider applications in cancer cases where either expression of antigen receptors on the cancer cells is sub-optimum or when cancer cells do not respond to the effects of immunotherapy, owing to the fact that in RIT cytotoxic effects are exerted by the radionuclide tagged with the antibody rather than the antibody itself.^8–11 The prime advantage of using radiolabeled antibodies in place of unlabeled ones for cancer treatment lies in the fact that by targeting a limited number of cancer cells in the tumor mass, a large number of such cells can be destroyed owing to the cross-fire effect exerted by the particulates (beta/alpha/Auger electron) generated due to the decay of the associated radionuclide.^3,6,7,12,13 Moreover, the amount of antibodies required for RIT is significantly lower compared to that needed for immunotherapeutic modality, as the role of antibodies in the former is just limited to shedding off the radiation load at the target site which in turn reduces the chemotoxic dose burden of the immunotherapy to a significant extent.⁶

The concept of using radiolabeled antibodies for the treatment of cancer is not new. However, initial attempts of using antibodies for RIT have not achieved the desired success which is primarily attributed to the use of antibodies of murine origin, as the corresponding radiolabeled agents suffered from serious shortcomings namely, short in vivo residence time, generation of human anti-mouse antibody response, etc.³ Recent advancements in genetic engineering leading to facile production of chimeric and humanized monoclonal antibodies have helped to overcome majority of such shortcomings and this has regenerated the interest in employing various unlabeled as well as radiolabeled monoclonal antibodies for the treatment of various types of human maladies.³ Trastuzumab (Herceptin®), a humanized monoclonal antibody, is the outcome of such efforts which led to its approval by the US-FDA (Food and Drug Administration of United States of America) for the treatment of patients suffering from metastatic breast cancer over-expressing human epidermal growth factor receptor 2 (HER2) antigen receptors. Though its use in the radiolabeled form is yet to receive US-FDA approval, clinical trials involving radiolabeled trastuzumab have been initiated in the recent past.⁸

For developing as a RIT agent for the treatment of HER2 receptor positive breast cancer patients, trastuzumab can be radiolabeled with a suitable particulate emitter, which decays by emission of either β⁻ or α particles or by release of Auger electrons.³ In the last decade, ¹⁷⁷Lu has emerged as one of the most useful radionuclides for the development of endoradiotherapeutic agents, particularly for the treatment involving small or medium-sized tumorous lesions.^8,14–16 Lutetium-177 decays to stable ¹⁷⁷Hf by release of β⁻ particles having a maximum energy of 497 keV along with emission of gamma photons of suitable energy [E_γ: 113 keV (6.4%), 208 keV (11%)] which helps in theranostic intervention enabling simultaneous scintigraphic and dosimetric evaluations.^14–16 The comparatively longer half-life (T_1/2 = 6.73 d) of the radionuclide helps in transporting ¹⁷⁷Lu or [¹⁷⁷Lu]Lu-based agents to the remote places from its production site without significant loss of activity due to radioactive decay.^14–16 Another advantage of using ¹⁷⁷Lu derives from its convenient and economic production with adequate specific activity and high radionuclidic purity using a medium flux reactor due to the large thermal neutron capture cross-section of ¹⁷⁶Lu (σ = 2100 b).^14–16

Direct incorporation of ¹⁷⁷Lu in the trastuzumab moiety is not viable owing to the non-availability of suitable functional groups in the antibody framework which are capable of forming stable complexes with the radiolanthanide.¹⁷ Therefore, in the present work, radiolabeling was mediated through a bi-functional chelating agent (BFCA), namely p-NCS-benzyl-DOTA [S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid]. It is well-documented that DOTA [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid] being a macrocyclic chelating agent forms thermodynamically stable complexes with radiolanthanides, such as ¹⁷⁷Lu (log K ∼ 25.4).^18–22 Moreover, radiolanthanide complexes of DOTA or its derivatives have superior in vivo kinetic inertness, which is one of the desired characteristics of any radiopharmaceutical, compared to their acyclic counterparts making DOTA derivatives preferred choices as BFCAs for radiolabeling with ¹⁷⁷Lu.^19,20

In spite of having serious potential, clinical studies with [¹⁷⁷Lu]Lu-trastuzumab are still in their infancy. One of the primary requirements for initiating widespread clinical evaluation of this agent is to develop a robust methodology which will enable formulation of this agent with a high degree of reproducibility in hospitals or central radiopharmacies. In the present work, attempts have been made to develop such a methodology which will enable formulation of [¹⁷⁷Lu]Lu-trastuzumab patient dose with a high radiochemical yield using reactor-produced carrier-added ¹⁷⁷Lu. Herein we report conjugation of trastuzumab with p-NCS-benzyl-DOTA, purification of the DOTA–trastuzumab conjugate and formulation of [¹⁷⁷Lu]Lu-trastuzumab patient dose by variation of various reaction parameters such as the ligand to metal ratio, pH, incubation time and selection of a suitable radio-protecting agent. We also report the in vitro evaluation of [¹⁷⁷Lu]Lu-trastuzumab in HER2 positive SK-OV-3 and SK-BR-3 as well as in triple negative MDA-MB-231 cancer cell lines and preliminary pharmacokinetic studies in a healthy Swiss mouse model. Herein, we also document preliminary clinical studies in patients suffering from breast cancer in order to ascertain the targeting efficacy of the radiolabeled preparation.

Experimental

Materials and methods

Trastuzumab (CANMAb™) was procured from Biocon (India). p-NCS-benzyl-DOTA was procured from Macrocyclics (USA). HEPES [(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)], sodium acetate, sodium carbonate and sodium bicarbonate used for the preparation of different buffer systems were procured from Sigma-Aldrich (USA). Ultra-centrifugal units (10 kDa MW cut off) required for purification of the antibody–BFCA conjugate were purchased from Merck Millipore Limited (Germany). PD-10 (Sephadex G-25 M) columns used for purification of the radiolabeled antibody complex were obtained from GE-Healthcare (India). Lutetium-177 was produced in-house following a procedure reported elsewhere^15,16 and obtained from the Radiochemicals section of Radiopharmaceuticals Division of our Institute [Bhabha Atomic Research Centre (BARC), Mumbai, India]. Non-radioactive LuCl₃ (99.99% chemically pure) used as a carrier was procured from Sigma-Aldrich. All other chemicals used in the present study were of analytical reagent (AR) grade and obtained from reputed local manufacturers.

MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) mass spectrometry (MS) was carried out using a MALDI-TOF mass spectrometer (UltrafleXtreme, Bruker, Germany). Whatman™ grade 3MM chromatography paper (cellulose fibers as the stationary phase) utilized for PC (paper chromatography) was purchased from Waters (USA). HPLC (high performance liquid chromatography) analyses were performed using a dual pump JASCO PU-2080 Plus HPLC system (Japan) employing a TSK-Gel G3000SWXL size-exclusion column (7.8 × 300 mm), which was pre-equilibrated with 0.05 M phosphate buffer (pH ∼ 6.8) maintaining a flow rate of 0.6 mL min⁻¹. The elution profile was monitored by detecting the radioactivity and UV (ultra-violet) signals (at 280 nm) using a Gina Star Radiometric NaI(Tl) detector (Raytest, Germany) and a JASCO 2075 Plus tunable absorption detector (Japan), respectively, coupled with the HPLC system. Durapore® polyvinylidene fluoride (PVDF) membrane filters (47 mm, 0.22 μm), used for filtration of solvents for HPLC, were procured from Merck Millipore. All radioactive counting was performed using a well-type NaI(Tl) scintillation detector, procured from Electronics Corporation of India Limited (India), unless mentioned otherwise. The baseline and window for the radioactive counting were adjusted to 150 keV and 100 keV, respectively, so as to utilize the 208 keV gamma photon emission of ¹⁷⁷Lu. An isotope dose calibrator used for measurement of radioactivity associated with patient dose formulation was obtained from Capintec (CRC-15 BETA, CII-capintec, USA).

Human ovarian cancer cell line SK-OV-3, human breast cancer cell line SK-BR-3 and triple-negative breast cancer cell line MDA-MB-231 were procured from the National Center for Cell Science (India). HER2 protein (recombinant, expressed in HEK293 cells) was procured from Sigma-Aldrich (USA). Animal experimentation was carried out in healthy Swiss mice which were bred and reared in the laboratory animal facility of Radiation Biology and Health Sciences Division of our Institute under standard management practice. Radioactive counting associated with the animal studies was carried out using a flat-type NaI(Tl) scintillation counter, procured from Electronics Corporation of India Limited (India), using the same counting set-up as mentioned above. The animal studies reported in the present article were approved by the Institutional Animal Ethics Committee (IAEC) of BARC and all animal experiments were carried out in strict compliance with the institutional (IAEC-BAEC) guidelines following the relevant national laws related to the conduct of animal experimentation (Prevention of Cruelty to Animals Act, 1960).

Clinical studies were performed in 8 female patients suffering from metastatic breast cancer over-expressing HER2 receptors. The studies were performed as per the established and approved safety guidelines, in line with the Declaration of Helsinki. Approval for clinical studies was obtained from the Institutional Medical Ethics Committee of the Tata Memorial Hospital (Mumbai) and informed written consent was obtained from the patients prior to the administration of the agent.

Preparation of the trastuzumab–p-NCS-benzyl-DOTA (DOTA–trastuzumab) conjugate

Trastuzumab–p-NCS-benzyl-DOTA (subsequently referred to as the DOTA–trastuzumab conjugate) was prepared by incubating trastuzumab (5 mg, 33.75 nmol) with 20 μL of p-NCS-benzyl-DOTA (337.5 nmol, 200 μg, 10 μg μL⁻¹) in sodium carbonate buffer (0.2 M, pH ∼ 9.5) at 37 °C for 17 h. For the conjugation reaction, trastuzumab and p-NCS-benzyl-DOTA were used in a molar ratio of 1 : 10. Post-incubation, buffer exchange as well as removal of non-conjugated p-NCS-benzyl-DOTA was carried out employing ultra-centrifugal filtration (9 cycles, 3800 rpm, 15 min) using 0.2 M HEPES buffer. The final volume of the purified DOTA–trastuzumab conjugate was made up to 500 μL by addition of 0.2 M HEPES buffer (pH ∼ 5).

Determination of the average number of p-NCS-benzyl-DOTA molecules attached per trastuzumab moiety

The purified DOTA–trastuzumab conjugate was analyzed using a MALDI-TOF MS for determination of the average number of p-NCS-benzyl-DOTA molecules attached per trastuzumab moiety. For mass analyses, samples of both trastuzumab and the purified DOTA–trastuzumab conjugate were dissolved in 0.2 M HEPES buffer (pH ∼ 5) and the concentration of the solutions was adjusted to 20 mg mL⁻¹ using ultra-centrifugal filtration units. Aliquots were drawn separately from each sample and analyzed by MALDI-TOF MS. The average number of p-NCS-benzyl-DOTA molecules attached per antibody moiety was calculated by dividing the difference in the molecular mass of the DOTA–trastuzumab conjugate and trastuzumab by the molecular mass of a single p-NCS-benzyl-DOTA unit.

Radiolabeling of the purified DOTA–trastuzumab conjugate with ¹⁷⁷Lu

The procedure for radiolabeling of the DOTA–trastuzumab conjugate with [¹⁷⁷Lu]LuCl₃ was optimized by varying various reaction parameters viz., the ligand to metal ratio, pH of the reaction mixture, and incubation period over an appreciable range.

Variation in the percentage radiochemical yield with the ligand to metal ratio

To arrive at the optimum ligand to metal ratio required for preparing [¹⁷⁷Lu]Lu-trastuzumab with a high radiochemical yield, the DOTA–trastuzumab conjugate (0.5 mg, 3.29 nmol) was dissolved in 0.2 M HEPES buffer (pH ∼ 5.0, 125 μL) and radiolabeled using a fixed amount of radioactive Lu i.e. [¹⁷⁷Lu]LuCl₃ [0.12 μg, 74 MBq (2 mCi)] along with variable amounts of non-radioactive Lu. For this, a stock solution of carrier Lu was prepared by dissolving non-radioactive LuCl₃ in Milli-Q water (2 mg/10 mL with respect to the content of Lu). Four different ligand to metal ratios viz. 1 : 1, 1 : 2, 1 : 3 and 1 : 4 [corresponding to 0.58 μg (3.29 nmol), 1.16 μg (6.59 nmol), 1.74 μg (9.87 nmol) and 2.32 μg (13.16 nmol) of Lu, respectively] were studied. The final pH of the reaction mixtures was adjusted to ∼5.6 using 0.2 M sodium acetate buffer (pH ∼ 5.6) prior to incubation at 37 °C for a period of 30 min. Post-radiolabeling, aliquots were withdrawn from each of the four reaction mixtures and separately analyzed for determining the percentage radiochemical yield following the quality control procedures mentioned below.

Variation in the percentage radiochemical yield with reaction pH

Attempts were also made to study the effect of variation of pH of the reaction mixture on the percentage complexation yield of [¹⁷⁷Lu]Lu-trastuzumab by carrying out the radiolabeling at four different pH values viz. 5.0, 5.6, 6.0 and 7.0. For adjustment of the reaction pH for the first two pH values (5.0 and 5.6), 0.2 M sodium acetate buffers of respective pH were used; whereas for the latter two values (6.0 and 7.0), 0.2 M sodium acetate solution and 0.05 N hydrochloric acid were used. All the reaction mixtures used for studying the effect of pH contained 0.5 mg of DOTA–trastuzumab conjugate (3.29 nmol) dissolved in 0.2 M HEPES buffer (125 μL, pH ∼ 5.0), [¹⁷⁷Lu]LuCl₃ [0.12 μg, 74 MBq (2 mCi)] and non-radioactive Lu [1.04 μg, 5.92 nmol] so as to maintain a ligand to metal ratio of 1 : 2. All the reaction mixtures were incubated at 37 °C for a period of 30 min. The percentage radiochemical yield was determined by the quality control procedures mentioned below.

Variation in the percentage radiochemical yield with the incubation period

For studying the effect of the incubation period on the percentage radiochemical yield of [¹⁷⁷Lu]Lu-trastuzumab, the DOTA–trastuzumab conjugate (0.5 mg, 3.29 nmol) dissolved in 0.2 M HEPES buffer (125 μL, pH ∼ 5.0) was incubated with [¹⁷⁷Lu]LuCl₃ [0.12 μg, 74 MBq (2 mCi)] in the presence of non-radioactive Lu [1.04 μg, 5.92 nmol] (so as to maintain a ligand to metal ratio of 1 : 2) at 37 °C for a period of 60 min. Aliquots were withdrawn from the reaction vial at four different incubation periods viz. 15, 30, 45 and 60 min and the corresponding percentage radiochemical yield of [¹⁷⁷Lu]Lu-trastuzumab was determined employing the quality control methods mentioned below.

Quality control of [¹⁷⁷Lu]Lu-trastuzumab

The percentage radiochemical yield of [¹⁷⁷Lu]Lu-trastuzumab was determined by PC as well as HPLC studies. PC analyses were carried out by spotting a small aliquot (∼5 μL) of the reaction mixture 1.5 cm above one of the ends of the PC strip (10 cm × 1 cm) and eluting the strip using 0.01 M sodium citrate buffer (pH ∼ 5.0) as the mobile phase. The strip was dried and cut into 1 cm segments. The radioactivity associated with each segment was determined using a well-type NaI(Tl) detector. On the other hand, HPLC analyses were performed by injecting a small aliquot of the reaction mixture into the HPLC column and running the chromatogram in an isocratic mode using 0.05 M phosphate buffer mixed with 0.05% NaN₃ as the mobile phase.

Effect of addition of radio-protectors on the percentage radiochemical yield of [¹⁷⁷Lu]Lu-trastuzumab

Two radio-protecting agents, namely gentisic acid and ascorbic acid, were used to study the effect of addition of such agents during preparation on the percentage radiochemical yield and stability of the [¹⁷⁷Lu]Lu-trastuzumab complex. Stock solutions of the radio-protecting agents were prepared by dissolving these agents separately in minimum volume of Milli-Q water and adjusting the pH of the solutions at ∼5.5 using 2 N NaOH solution. Three different radiolabeling reactions were set-up in order to have a comparative evaluation of the effect of addition of radio-protecting agents. In the first two reaction mixtures, ascorbic acid (5 mg, 0.03 mmol, 100 μL) or gentisic acid (5 mg, 0.03 mmol, 100 μL) was added to the DOTA–trastuzumab conjugate (0.5 mg, 3.29 nmol) dissolved in 0.2 M HEPES buffer (125 μL) prior to the addition of [¹⁷⁷Lu]LuCl₃ [0.12 μg, 74 MBq (2 mCi)] and non-radioactive Lu [1.04 μg, 5.92 nmol] (so as to maintain a ligand to metal ratio of 1 : 2). In the third reaction mixture, no radio-protecting agent was added, but the rest of the ingredients were kept identical. All three reaction mixtures were incubated at 37 °C for 30 min. Aliquots were withdrawn from each reaction mixture and analyzed for determining the percentage radiochemical yield following the quality control methods mentioned above. The reaction mixtures were subsequently stored at room temperature till 7 d post-preparation in order to study the in vitro stability patterns of the preparations. For this, aliquots were separately withdrawn from each of the reaction mixtures at different post-preparation time points (1, 3 and 7 d) and analyzed for determining the percentage radiochemical yield following the quality control processes described above.

Preparation of patient dose of [¹⁷⁷Lu]Lu-trastuzumab

The optimized protocol for preparation of [¹⁷⁷Lu]Lu-trastuzumab was deduced based on the results of the above-mentioned experiments and was utilized for the preparation of the patient dose of the radiolabeled agent. For the preparation of the patient dose of [¹⁷⁷Lu]Lu-trastuzumab, the purified DOTA–trastuzumab conjugate (3.0 mg, 19.79 nmol) was dissolved in 0.2 M HEPES buffer (0.3 mL, pH ∼ 5) and incubated with [¹⁷⁷Lu]LuCl₃ [39.58 nmol (7.0 μg), 3.90 GBq (∼105 mCi)] in 0.3 mL of 0.2 M sodium acetate buffer (pH ∼ 5.6) in the presence of ∼0.1 mL pH adjusted (to ∼5.5 using 2 N NaOH solution) aqueous ascorbic acid (10 mg, 0.06 mmol) at 37 °C for 30 min. Post-labeling, aliquots were withdrawn from the reaction mixture and the percentage radiochemical yield of [¹⁷⁷Lu]Lu-trastuzumab was determined by PC as well as HPLC studies following the protocol mentioned above.

Purification of patient dose of [¹⁷⁷Lu]Lu-trastuzumab

Studies were also carried out in order to standardize the method of purification of the [¹⁷⁷Lu]Lu-trastuzumab patient dose, so as to improve the percentage radiochemical yield of the radiolabeled agent, if required. Purification of [¹⁷⁷Lu]Lu-trastuzumab was achieved using PD-10 desalting columns following the procedure mentioned below. Prior to purification, a PD-10 column was equilibrated by passing 25 mL of 0.2 M sodium acetate buffer of pH ∼ 5.6. Post-equilibration, the reaction mixture was loaded onto the column and eluted using the same buffer system. The eluate was collected as 1.0 mL fractions and 8 such fractions were collected. The fraction containing majority of [¹⁷⁷Lu]Lu-trastuzumab was identified by HPLC analyses. The percentage recovery of the radiolabeled product after purification was calculated by measuring the radioactivity collected in the fraction containing the agent after purification and the total radioactivity loaded on the column before purification, which were determined using a pre-calibrated isotope dose calibrator.

In vitro stability studies

The in vitro stability of [¹⁷⁷Lu]Lu-trastuzumab was studied at different post-preparation time points viz. 1, 4, 5 and 7 d by storing the radiolabeled preparation in three different media viz. PBS (phosphate buffered saline), human serum and PBS with EDTA (ethylenediaminetetraacetic acid, 50 molar excess with respect to Lu utilized for this study) at room temperature. Aliquots (10 μL) were withdrawn from the radiolabeled preparation at the respective post-preparation time points and were analyzed via PC as well as HPLC following the quality control procedures mentioned earlier.

In vitro cell binding studies

Human ovarian cancer cells SK-OV-3 and human breast cancer cells SK-BR-3 were grown to 70–80% confluence in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere containing 5% CO₂ at 37 °C. MDA-MB-231 (triple negative breast cancer) cells, used as a negative control, were also grown under identical conditions. For in vitro cell binding studies, ∼1 × 10⁶ cells were seeded in 12-well tissue culture plates and incubated overnight at 37 °C. The adherent cells were subsequently incubated with [¹⁷⁷Lu]Lu-trastuzumab [50 nM, 5 MBq (135 μCi)] for a period of 2 h. Subsequently, the cells were washed twice with ice-cold 0.05 M PBS (pH ∼ 7.4) and solubilized with 1 N aqueous NaOH solution (1 mL). Finally the solution was counted in a NaI(Tl) gamma counter to determine the radioactivity associated with cells. Assays were carried out in triplicate. The percentage radioactivity bound to the cells with respect to the total radioactivity added was determined and expressed as mean ± standard deviation (SD). Inhibition assays were also performed under identical conditions by incubating the cancer cells with [¹⁷⁷Lu]Lu-trastuzumab [50 nM, 5 MBq (135 μCi)] in the presence of excess unlabeled trastuzumab (3.3 μM).

Cell internalization assay

Internalization experiments were performed with SK-OV-3 cells. Cells (∼1 × 10⁶) were seeded in 24-well cell culture plates one day prior to the experiment. The cells were incubated with [¹⁷⁷Lu]Lu-trastuzumab [50 nM, 5 MBq (135 μCi)] for 24 h at 37 °C in a humidified atmosphere containing 5% CO₂. Post-incubation, the supernatant was removed and the cells were washed with ice-cold PBS. To remove surface-bound radioactivity, the cells were incubated twice with 0.5 mL sodium acetate buffer (0.1 M glycine, pH ∼ 2.5) for 1 min. The cells were then washed with 1.0 mL of ice-cold PBS and lysed using 0.5 mL of 1 N NaOH. The internalized fraction of the radioactivity was measured using the NaI(Tl) counter.

Saturation binding assay

For the saturation binding assay, SK-OV-3 cells (∼1 × 10⁶) were seeded in a 12-well plate. [¹⁷⁷Lu]Lu-trastuzumab solution was prepared in PBS (containing 0.1% BSA) with a gradient concentration from 7.5–120 nM. After adding the antibody solutions to each well, the cells were incubated at 4 °C for 2 h. The non-specific binding values were obtained by co-incubating the cells with 100 fold excess unlabeled trastuzumab. At the end of the incubation period, the cells were washed with PBS twice and harvested for gamma counting analysis. Based on the overall bound counts obtained after subtracting counts corresponding to non-specific bindings with SK-OV-3 cells, the K_d and β_max values were determined. The HER2 receptor density on SK-OV-3 cells was determined using GraphPad Prism software.

Determination of the immunoreactive fraction (IRF) of [¹⁷⁷Lu]Lu-trastuzumab

The immunoreactivity of the ¹⁷⁷Lu[Lu]-DOTA–trastuzumab preparations was assessed by the Lindmo method,²³ which extrapolates the binding of the radiolabeled antibody at an infinite excess antigen. SK-OV-3 cells (6.25 × 10⁴–1 × 10⁷) were harvested in 900 μL DMEM media (in triplicate). A fixed amount of ¹⁷⁷Lu[Lu]-trastuzumab (∼5000 cps) was added to each tube containing cells and incubated at 4 °C for about 2 h with gentle shaking. For estimating the non-specific binding, inhibition studies were carried out by addition of excess unlabeled DOTA–trastuzumab conjugate. Cells were washed twice with cold PBS and radioactivity associated with cells was measured using the NaI(Tl) counter. The obtained data were subsequently plotted as the reciprocal of the cell concentration (X-axis) against the reciprocal of the bound fraction (Y-axis) and eventually fitted according to a linear regression method using GraphPad Prism software. The IRF was calculated using the equation {100 × (1/(Y-intercept))} from the graph. The experiment was carried out in triplicate and repeated thrice.

Determination of the affinity constant of [¹⁷⁷Lu]Lu-trastuzumab towards pure HER2 protein

The affinity of DOTA conjugated trastuzumab was determined using commercially available purified human epidermal growth factor receptor 2. HER2 protein (50 ng) in phosphate buffer (200 μL) containing Ca²⁺ and Mg²⁺ was added to the wells of a 96-well plate and incubated overnight at 4 °C for coating the wells. The solution was removed from the wells followed by addition of ∼150 μL of 1% bovine serum albumin (BSA) in PBS. The plate was incubated for 1 h at room temperature and the BSA solution was removed. Various dilutions of [¹⁷⁷Lu]Lu-trastuzumab (6.5–65 nM) were added to the wells. After addition of [¹⁷⁷Lu]Lu-trastuzumab, the plates were incubated for 2 h at 37 °C. The wells were washed with PBS twice after incubation. To count the radioactivity, 0.2 N NaOH was added to the wells and counted for cell associated radioactivity. The data were analyzed with a least squares non-linear regression method (GraphPad Prism software) to determine the K_d and a Scatchard transformation was performed.

Bio-evaluation in a healthy Swiss mouse model

The pharmacokinetic behavior of [¹⁷⁷Lu]Lu-trastuzumab was evaluated by carrying out bio-distribution studies in a healthy Swiss mouse model at four different post-administration time points viz. 1, 2, 5 and 7 d. [¹⁷⁷Lu]Lu-trastuzumab, prepared under the optimized conditions, was diluted using sterile normal saline and administered [150 μL, ∼100 μCi (3.7 MBq)] in each experimental animal through one of the lateral tail veins. The animals were kept at the normal laboratory atmosphere with adequate supply of food and water post-injection. The animals were sacrificed by CO₂ asphyxiation and three animals were utilized for each time point. The animals were sacrificed at the designated post-administration time. Blood was withdrawn from each animal by cardiac puncture immediately after sacrificing the animals. Subsequently, the animals were dissected and various organs/tissues were excised and weighed using a simple weighing balance. The activity associated with various organs/tissues was determined by counting the respective organ/tissue in a flat-type NaI(Tl) counter. The percentage of injected activity (% IA) accumulated in various organs/tissues was calculated from the above data and expressed as % IA per gram organ/tissue. The total activity accumulated in blood, skeleton and muscles was calculated by considering that 7%, 10% and 40% of the animal body weight are constituted by these organs/tissues, respectively.^24,25 The activity excreted through urine was indirectly calculated by subtracting the total activity accounted in all the organs/tissues from the total administered activity.

Clinical studies

Clinical administration, for studying the biological distribution of [¹⁷⁷Lu]Lu-trastuzumab, was carried out in 8 female patients suffering from immunohistochemically proven ER-negative, PR-negative, c-erbB2/HER2-positive metastatic breast cancer. All the patients underwent [¹⁸F]FDG (2-deoxy-2-¹⁸F-fluoroglucose) whole-body PET-CT (Positron emission tomography-computed tomography) scans prior to the administration of [¹⁷⁷Lu]Lu-trastuzumab. On average, 110 ± 14.5 MBq (2.97 ± 0.39 mCi) of [¹⁷⁷Lu]Lu-trastuzumab formulation [94.35–129.50 MBq (2.55–3.50 mCi)] was administered intravenously in each patient and serial scintigraphic images were recorded at various post-administration time points viz. 3, 24, 48, 72 and 120 h.

Results

Preparation of the DOTA–trastuzumab conjugate

Trastuzumab was conjugated to p-NCS-benzyl-DOTA in order to facilitate radiolabeling with ¹⁷⁷Lu (Fig. 1). A ligand to BFCA ratio of 1 : 10 was chosen for carrying out the coupling reaction based on our previous experience related to the optimization of the antibody to BFCA ratio while working with another monoclonal antibody namely, Rituximab.²⁶ Purification of the BFCA–antibody conjugate was carried out by ultra-centrifugal filtration whereby free or unconjugated p-NCS-benzyl-DOTA moieties were removed from the reaction mixture. The purified DOTA–trastuzumab conjugate, thus obtained, was characterized by HPLC where a single peak corresponding to the conjugate was observed indicating the absence of free/unconjugated BFCA (Fig. S1, ESI†). Determination of the average number of BFCA attached per trastuzumab molecule was carried out using MALDI-TOF mass spectrometry where a mass peak corresponding to trastuzumab was observed at 1481 12.234, while a mass peak corresponding to the DOTA–trastuzumab conjugate was observed at 151 531.312 (Fig. 2). Upon dividing the mass difference of the DOTA–trastuzumab conjugate and trastuzumab by the molecular mass of a single BFCA unit i.e. p-NCS-benzyl-DOTA (551.880), the value for the average number of BFCAs attached per trastuzumab moiety was found to be 6.15 ± 0.92.

Fig. 1. Schematic representation of conjugation and complexation reactions leading to formation of [¹⁷⁷Lu]Lu-trastuzumab.

Fig. 2. Mass spectra of (a) trastuzumab and (b) the DOTA–trastuzumab conjugate.

Preparation of [¹⁷⁷Lu]Lu-trastuzumab

For arriving at an optimized protocol for the preparation of [¹⁷⁷Lu]Lu-trastuzumab various reaction parameters viz. the ligand to metal ratio, reaction time, and reaction pH were varied extensively and the effect of addition of two different radio-protecting agents on the complexation yield was studied. The incubation temperature for all the experiments was fixed at 37 °C due to inevitable limitation of using higher temperatures leading to the possible structural damage and denaturation of the antibody. Variation of the ligand to metal ratio was attempted in order to find out the maximum ¹⁷⁷Lu activity that can be loaded in a given amount of DOTA–trastuzumab conjugate without compromising the percentage radiochemical yield. Four different ligand to metal ratios, starting from 1 : 1 to 1 : 4 were used for complexation and it was observed that the [¹⁷⁷Lu]Lu-trastuzumab complex can be prepared with >90% radiochemical yield using all the four ligand to metal ratios. However, considering the availability of ¹⁷⁷Lu with a specific activity of around 555–740 GBq mg⁻¹ (15–20 Ci mg⁻¹) at our end, it was decided to use a ligand to metal ratio of 1 : 2, which was sufficient to obtain a patient dose of 1.85 GBq (50 mCi) without compromising the percentage radiochemical yield of the preparation.

In order to study the effect of the pH of the reaction mixture on the percentage radiochemical yield, radiolabeling studies were carried out at four different reaction pH values between 5.0 and 7.0. However, no significant differences were observed in the percentage radiochemical yield of the [¹⁷⁷Lu]Lu-trastuzumab complex in any of the reaction pH (p > 0.05) (Fig. S2, ESI†). For the present work, pH 5.6 was chosen as the optimized radiolabeling pH, as this is the highest pH of sodium acetate buffer which can be achieved without extraneous addition of any acid or base. Studies related to the variation in incubation period revealed that there was a marginal increase in the percentage radiolabeling yield (from 91.90 ± 2.90 to 95.39 ± 1.65) when the period of incubation was increased from 15 min to 30 min. However, no significant change in the percentage radiochemical yield (94.66 ± 1.88) was observed on further increasing the incubation period up to 60 min. Hence, 30 min was chosen as the optimum incubation period for preparation of the [¹⁷⁷Lu]Lu-trastuzumab complex (Fig. S3, ESI†).

The effect of using two different radio-protecting agents, namely ascorbic acid and gentisic acid, was studied and the study revealed that the use of ascorbic acid had the least effect on the percentage radiochemical yield of the [¹⁷⁷Lu]Lu-trastuzumab complex. Therefore, the use of ascorbic acid as a radio-protecting agent was incorporated in the optimized radiolabeling protocol for the preparation of the [¹⁷⁷Lu]Lu-trastuzumab complex. The HPLC profiles depicting the stability pattern of [¹⁷⁷Lu]Lu-trastuzumab prepared without adding any radio-protecting agent (Fig. S4, ESI†) as well as prepared using ascorbic acid (Fig. S5, ESI†) or gentisic acid (Fig. S6, ESI†) at various post-preparation time-points (the same day of preparation, 1, 3 and 7 d) are available in the ESI.†

Preparation of patient dose of [¹⁷⁷Lu]Lu-trastuzumab

The optimized protocol used for the preparation of patient dose of [¹⁷⁷Lu]Lu-trastuzumab resulted in the formulation of the agent with an average percentage radiochemical yield of 91.08 ± 2.54. Since it is desirable that the radiochemical purity (RCP) for any therapeutic radiopharmaceutical should be ≥95%, attempts were made to further improve the radiochemical purity of the preparation by using a PD-10 desalting column. This additional step improved the radiochemical purity of the formulation to the desirable range i.e. >95% as is evident from the PC (R_f for [¹⁷⁷Lu]Lu-trastuzumab 0.0–0.1; R_f for free [¹⁷⁷Lu]LuCl₃ 0.9–1.0) (Fig. S7, ESI†) and HPLC studies (R_t for [¹⁷⁷Lu]Lu-trastuzumab = 15.5 ± 0.5 min; R_t for free [¹⁷⁷Lu]Lu(iii) = 21.5 ± 0.85) (Fig. 3). However, passing the [¹⁷⁷Lu]Lu-trastuzumab preparation through the PD-10 column was associated with the loss of radioactivity due to the trapping of activity in the column matrix as well as in the loading vial. On average, (72.8 ± 1.2)% of the loaded activity could be retrieved after the purification through the PD-10 column.

Fig. 3. HPLC chromatograms: (a) UV profile and (b) radioactive profile of [¹⁷⁷Lu]Lu-trastuzumab (monitored by detecting the UV and radioactivity signals using UV and NaI(Tl) detectors coupled with the HPLC system, respectively).

The purified preparation of [¹⁷⁷Lu]Lu-trastuzumab was finally subjected to Millipore filtrations (0.22 μm) prior to the administration in patients to ensure the sterility of the product. The final product could be prepared with a molar activity (A_m) of 108.04–144.30 GBq μmol⁻¹ (2.92–3.90 Ci μmol⁻¹) after accounting for the losses of radioactivity during its preparation, purification and filtration.

In vitro stability studies

Owing to the long half-life of ¹⁷⁷Lu and longer blood circulation periods of antibodies, it was felt pertinent to monitor the in vitro stability pattern of [¹⁷⁷Lu]Lu-trastuzumab in different storage media. The study revealed a slow and gradual decrease of the percentage radiochemical purity of [¹⁷⁷Lu]Lu-trastuzumab with the progress of time. The radiolabeled agent maintained a percentage radiochemical purity of 91.78 ± 2.77, 90.01 ± 1.10 and 91.34 ± 2.66 at 1 d post-preparation, which gradually reduced to 82.25 ± 3.62, 80.79 ± 1.09 and 79.43 ± 1.12 at 7 d post-preparation when stored at room temperature in PBS, human blood serum and PBS containing EDTA in 50 times molar excess of Lu, respectively (Fig. S8, ESI†).

In vitro cell studies

In vitro cell binding studies of [¹⁷⁷Lu]Lu-trastuzumab were carried out in HER2 over-expressing SK-OV-3 and SK-BR-3 cell lines. The agent exhibited a binding of (32.3 ± 7.2)% in SK-BR-3 cells which decreased to (9.6 ± 3.0)% when co-incubated with an excess (3.3 μM) of unlabeled trastuzumab. When the cell binding studies were carried out with SK-OV-3 cells, the agent exhibited a maximum binding of (17.42 ± 1.33)% which showed a significant decrease to (6.50 ± 0.53)% when co-incubated with an excess (3.3 μM) of unlabeled trastuzumab. The decrease in binding after co-incubation with excess unlabeled trastuzumab observed in both cases indicated the specificity of the radiolabeled preparation towards the HER2 receptors. To further confirm the specificity of [¹⁷⁷Lu]Lu-trastuzumab towards HER2 receptors as well as to determine the extent of non-specific binding, cell binding experiments were also performed in triple negative MDA-MB-231 cells, where the agent exhibited binding and inhibition values of (4.43 ± 0.76)% and (1.96 ± 0.66)%, respectively. The results of the cell binding studies of [¹⁷⁷Lu]Lu-trastuzumab carried out in three different cell lines are shown in Fig. 4.

Fig. 4. Results of cell binding studies of [¹⁷⁷Lu]Lu-trastuzumab in HER2 positive cell lines viz. SK-Br-3 and SK-OV-3 and triple negative MDA-MB-231 cell lines (* indicates reduced values of percentage cell binding upon addition of unlabeled trastuzumab).

The internalization assay in SK-OV-3 cells revealed that [¹⁷⁷Lu]Lu-trastuzumab internalizes inside the cells as soon as the cells get exposed to the formulation. The cellular associated activity after 1 h of incubation was found to be (12.16 ± 0.60)% which further increased to (41.66 ± 1.52)% after 24 h of incubation. The results of the internalization assay of [¹⁷⁷Lu]Lu-trastuzumab in SK-OV-3 cell line are shown in Fig. 5.

Fig. 5. Graphical representation of the internalization assay of [¹⁷⁷Lu]Lu-trastuzumab in the SK-OV-3 cell line.

High binding affinity between the monoclonal antibody and the target antigen is another pre-requisite which must be fulfilled for the successful application of any radiolabeled antibody for the radiotherapeutic intervention of the disease. The binding affinity studies carried out in SK-OV-3 cells indicated that [¹⁷⁷Lu]Lu-trastuzumab exhibited a nanomolar level binding affinity (K_d = 13.61 nM) with a β_max value of 31.09 pmoles per 10⁶ cells. The β_max value was converted to obtain the concentration of antigens or receptors per cell and found to be 3.74 × 10⁴ (Fig. 6).

Fig. 6. Saturation binding assay and Scatchard plot for [¹⁷⁷Lu]Lu-trastuzumab depicting K_d and β_max values.

The immunoreactive fraction of [¹⁷⁷Lu]Lu-trastuzumab in SK-OV-3 cells was determined using the equation {100 × (1/(Y-intercept))} from the graph (Fig. 7) and was found to be (76.92 ± 2.80)% indicating appreciable retention of the bio-active fraction in the radiolabeled preparation.

Fig. 7. Graphical depiction of variation of total/bound activity versus inverse of cell concentration utilized to determine the percentage immunoreactive fraction of [¹⁷⁷Lu]Lu-trastuzumab.

Determination of the affinity constant of [¹⁷⁷Lu]Lu-trastuzumab towards pure HER2 protein

The formulation showed strong affinity towards HER2 receptor protein exhibiting a dissociation constant (K_d) value of 11.36 nM which was observed to be in agreement with the value obtained when the similar study was conducted in cell culture (SK-OV-3) (Fig. S9, ESI†).

Bio-evaluation studies

Bio-evaluation involving biological distribution and pharmacokinetic evaluation studies of [¹⁷⁷Lu]Lu-trastuzumab was carried out in a normal healthy small animal model and the results of the biodistribution studies are tabulated in Table 1. The studies revealed high uptake and longer retention of radioactivity in blood [(19.54 ± 5.09), (18.35 ± 2.00), (14.15 ± 4.34) and (11.48 ± 3.24)% IA per g at 1, 2, 5 and 7 d post-administration, respectively]. High non-specific uptake of the radiolabeled antibody was also observed in various organs such as the liver, gastrointestinal tract (GIT) and muscles at the initial post-administration time point [(9.27 ± 3.19), (2.85 ± 0.06) and (1.12 ± 0.10)% IA per g, respectively at 1 d post-administration]. However, gradual but slow clearance of radioactivity was observed from all these non-target organs with the progress of time [(5.43 ± 1.16), (0.88 ± 0.04) and (0.82 ± 0.23)% IA per g, respectively at 7 d post-administration]. Low uptake of the radiotracer observed in the kidneys [(6.06 ± 2.84), (8.79 ± 1.38), (5.77 ± 1.29) and (5.24 ± 2.01)% IA per g at 1, 2, 5 and 7 d post-administration, respectively] as well as slow urinary excretion [(36.72 ± 13.83), (46.16 ± 1.76), (53.14 ± 14.28) and (64.64 ± 13.50)% IA at 1, 2, 5 and 7 d post-administration, respectively] indicated significant clearance of the administered radioactivity through the hepatobiliary pathway. This observation is in concordance with the fact that high molecular weight compounds such as antibodies exceed the threshold of glomerular filtration which impedes their excretion through the renal pathway.

Bio-distribution pattern of [¹⁷⁷Lu]Lu-trastuzumab in normal Swiss mice.

Organ	% injected activity per gram (% IA per g) of organ/tissue
	1 day	2 days	5 days	7 days
Blood	19.54 ± 5.09	18.35 ± 2.00	14.15 ± 4.34	11.48 ± 3.24
Lungs	11.83 ± 5.17	10.41 ± 2.05	7.12 ± 2.19	8.14 ± 2.44
Heart	5.93 ± 0.64	4.63 ± 0.59	4.09 ± 1.02	3.71 ± 1.31
Stomach	1.09 ± 0.18	1.03 ± 0.04	1.10 ± 0.06	1.12 ± 0.22
GIT	2.85 ± 0.06	2.41 ± 0.02	1.95 ± 0.11	0.88 ± 0.04
Liver	9.27 ± 3.19	8.69 ± 0.84	10.27 ± 3.46	5.43 ± 1.16
Spleen	2.66 ± 0.57	4.33 ± 0.68	3.50 ± 1.03	2.66 ± 0.80
Kidneys	6.06 ± 2.84	8.79 ± 1.38	5.77 ± 1.29	5.24 ± 2.01
Muscle	1.12 ± 0.10	1.35 ± 0.26	0.87 ± 0.17	0.82 ± 0.23
Excretion	36.72 ± 13.83	46.16 ± 1.76	53.14 ± 14.28	64.64 ± 13.50

Clinical studies

The preliminary clinical evaluation of [¹⁷⁷Lu]Lu-trastuzumab in patients suffering from breast cancer over-expressing HER2 receptors revealed the target specificity of the radiolabeled preparation. Additionally, the cancerous lesions picked up by the agent were found to be in concordance with those found in the diagnostic PET scans, recorded with [¹⁸F]FDG in the same patient prior to the administration of [¹⁷⁷Lu]Lu-trastuzumab. As antibodies exhibit slower pharmacokinetics leading to higher uptake as well as longer retention in blood, it resulted in delayed enhancement of the tumor to background ratio. Consequently, whole-body scans of the patients exhibited the best tumor to background contrast only at 72 h post-administration. Fig. 8(a) shows the typical [¹⁸F]FDG PET-MIP (MIP: maximum intensity projection) and PET-CT scans of a cancer patient with metastatic breast lesions in the right breast, whereas Fig. 8(b) and (c) depict the whole-body scans of the same patient acquired after administration of [¹⁷⁷Lu]Lu-trastuzumab at two different post-administration time points viz. 48 and 72 h, respectively. The images clearly demonstrate the preferential accumulation of [¹⁷⁷Lu]Lu-trastuzumab in the breast lesions, which is in concordance with the [¹⁸F]FDG PET scans indicating the theranostic potential of the agent.

Fig. 8. (a) [¹⁸F]FDG (MIP)-PET-CT scans of a cancer patient suffering from metastatic breast cancer; (b) and (c) SPECT (anterior and posterior) scans of the same patient obtained using [¹⁷⁷Lu]Lu-trastuzumab at two different post-administration time points viz. 48 and 72 h, respectively.

Discussion

Breast cancer is one of the most common cancers encountered by women all over the world and causes the greatest number of cancer-related deaths among women.²⁷ It is documented that one-fourth of breast cancer patients over-express HER2 receptors and such patients are usually associated with poor clinical outcome.²⁸ The front-line treatment modalities employed for the treatment of breast cancer patients such as surgery, chemotherapy and radiotherapy sometimes produce sub-optimal results, particularly at the advanced stage of the disease. RIT involving radiolabeled trastuzumab may be useful for providing systemic therapy to such patients.⁵ Trastuzumab (Herceptin®) is a US-FDA approved drug, which is often used for the treatment of metastatic breast cancer.⁵ However, the associated cytotoxicity, particularly cardiotoxicity, often limits the use of Herceptin in breast cancer patients.²⁹ Moreover, Herceptin being an expensive antibody increases the cost of patient care to a significant extent. In this context, the use of [¹⁷⁷Lu]Lu-trastuzumab appears advantageous, as the therapy can be executed by using a considerably smaller amount of the antibody thus effectively reducing both the chemotoxic burden and cost associated with the treatment regime.

The formulation of patient dose of radiolabeled antibodies is quite challenging, primarily arising from the possibility of denaturation involving breakage of many of the weak linkages or bonds present in the antibody structure and thus destroying the highly ordered structure of the antibodies in their native state resulting in the loss of their bio-reactivity.³⁰ Therefore, successful formulation of radiolabeled antibodies requires careful consideration of several radiolabeling parameters such as the pH range at which radiolabeling can be performed, maximum temperature at which the reaction mixture can be incubated, maximum salt concentration, etc.³⁰

In the present study, efforts were predominantly directed towards standardizing a protocol for the preparation of [¹⁷⁷Lu]Lu-trastuzumab with high reproducibility, as the not-so-convenient formulation methodology and non-reproducible radiochemical yields limit the wider application of any radiolabeled product and cause impediment towards its clinical translation. Although a limited number of reports on preparation of [¹⁷⁷Lu]Lu-trastuzumab using different BFCAs are available in the contemporary literature,^4,8,11 detailed radiochemistry aspects related to the preparation of the agent are conspicuously not available. Therefore, during the present study, attempts have been directed to formulate the optimized radiolabeling protocol after studying the effect of variation of different reaction parameters with considerable details. Additionally, the authors have also attempted to make a head to head comparison of the conjugation procedure and radiochemistry parameters utilized in the present study with those used in the studies on ¹⁷⁷Lu[Lu]-trastuzumab formulation published elsewhere (Table 2).^4,5,8,11 A careful comparison amongst the agents prepared using DOTA-based chelators reveals that the present method requires reduced incubation period for radiolabeling (30 min) and results in the formation of the final product with a relatively higher specific activity (0.70–1.02 MBq μg⁻¹). Our present study provides relatively more emphasis on the radiochemistry part by documenting the systemic data involving the explicit details about the procedures leading to the optimized reaction protocol which was eventually utilized for scaling-up of the activity for the preparation of actual patient dose of ¹⁷⁷Lu[Lu]-trastuzumab, information not reported in similar reports published elsewhere.^4,5,8,11

Comparative evaluation of reaction parameters and results of the present study with the previously published literature studies on ¹⁷⁷Lu[Lu]-trastuzumab.

Agent	[177Lu]Lu-DOTA–trastuzumab (present study)	[177Lu]Lu-DTPA–trastuzumab (Kameswaran et al.)11	[177Lu]Lu-DOTA–trastuzumab (Bhusari et al.)5	[177Lu]Lu-DOTA–trastuzumab (Rasaneh et al.)4	[177Lu]Lu-DTPA–trastuzumab (Ray et al.)8
BFCA	p-NCS-benzyl-DOTA	p-NCS-benzyl-CHX-A′′-DTPA	DOTA-NHS ester	DOTA-NHS ester	CHX-A′′-DTPA
Linker					Not-mentioned
Ligand to BFCA ratio	1 : 10	1 : 10	1 : 20	1 : 20	1 : 10
Average number of BFCA attached per antibody	6.15 ± 0.92	3.0–4.0	7.2	5.7–6.0	1.7
177Lu	Carrier added ([177Lu]LuCl3)	Carrier added ([177Lu]LuCl3)	Carrier added ([177Lu]LuCl3)	Carrier added ([177Lu]LuCl3)	Not mentioned
pH	5.6	6.0	5.5	5.0	Final pH not reported
Incubation temperature	37 °C	Room temperature	37 °C	37 °C	37 °C
Time of incubation	30 min	15 min	120 min	180 min	120 min
% RCY & a% RCP	91.08 ± 2.54	Not reported	∼91	68 ± 1.7	Not reported
	>95	>95	>95	94 ± 0.7	>95
Specific activity	0.70–1.02 MBq μg−1	0.62 MBq μg−1	0.22–0.49 MBq μg−1	Not reported	1.39 MBq μg−1
Quality control	PC/HPLC	PC/HPLC	ITLC	ITLC	HPLC
Animal model	Healthy Swiss mice	Healthy Swiss mice	Healthy rats	Tumor bearing BALB/c mice	Tumor bearing athymic (nu/nu) mice
Clinical studies/outcome	Imaging studies indicated accumulation in IHC proven HER2 positive cancerous lesions	Not reported	Imaging studies indicated accumulation in IHC proven HER2 positive cancerous lesions	Not reported	Not reported

^aValue obtained after purification. IHC: immunohistochemically.

The use of the ligand to metal ratio of 1 : 2 was found to be sufficient for the formulation of a single patient dose of 1.85 GBq (50 mCi) using the medium specific activity ¹⁷⁷Lu available at our end without compromising the percentage radiochemical yield of the preparation. The [¹⁷⁷Lu]Lu-trastuzumab patient dose was prepared within 30 min of incubation at 37 °C with >90% radiochemical purity which was further improved to >95% by using a PD-10 desalting column. The radiolabeled preparation showed adequate in vitro stability in buffer, human blood serum and in the presence of EDTA. Evaluation of the radiolabeled preparation in HER2 positive cancer cell lines, namely SK-BR-3 and SK-OV-3, revealed the retention of efficacy of [¹⁷⁷Lu]Lu-trastuzumab prepared towards the target receptors. In fact, observation of a K_d value as low as 13.61 nM and an immunoreactive fraction of (76.92 ± 2.80)% indicated that the radiolabeling procedure used for the preparation of [¹⁷⁷Lu]Lu-trastuzumab has not significantly altered the affinity of the agent towards the target receptors.^31,32 The value of the dissociation constant (K_d) of 11.36 nM exhibited by [¹⁷⁷Lu]Lu-trastuzumab in HER2 protein, similar to that observed in HER2 positive cancer cell lines, further supplements the aforementioned observation.

It is worthwhile to mention that although preparation and pre-clinical evaluation of ¹⁷⁷Lu[Lu]-trastuzumab in animal models have been reported elsewhere,^4,8,11 there are hardly any reports documenting the administration of ¹⁷⁷Lu[Lu]-trastuzumab in humans, aside from the article by Bhusari et al.⁵ A comparison of the bio-distribution data of ¹⁷⁷Lu[Lu]-trastuzumab in mouse models reported in the literature^4,8,11 with the present study shows that the agent prepared using DOTA derivatives as chelators exhibited relatively higher blood uptake and slower clearance compared to those agents prepared using DTPA-based derivatives (Table 3).^8,11 This may probably be due to the relatively more hydrophilic nature of DTPA-based derivatives compared to those prepared using the DOTA macrocyclic unit.³³

Comparative uptake of ¹⁷⁷Lu[Lu]-trastuzumab in blood and liver of mice at 24 h post-administration (present study vs. the previously published literature studies on ¹⁷⁷Lu[Lu]-trastuzumab).

Agent	[177Lu]Lu-DOTA–trastuzumab (present study)	[177Lu]Lu-DTPA–trastuzumab (Kameswaran et al.)11	[177Lu]Lu-DOTA–trastuzumab (Rasaneh et al.)4	[177Lu]Lu-DTPA–trastuzumab (Ray et al.)8
Uptake in blood (in% IA/g)	19.54 ± 5.09	13.5 ± 0.5	24.76 ± 4.31	12.68 ± 3.08
Uptake in liver (in% IA/g)	9.27 ± 3.19	7.9 ± 1.2	6.02 ± 0.92	7.80 ± 2.52

As far as the effect of procedural variations adopted to prepare the radiolabeled agent on clinical outcome is concerned, both studies, the present and that reported by Bhusari et al.,⁵ have been carried out using sub-therapeutic doses of the radiolabeled agent and thus at this stage it is premature to comment on the therapeutic efficacy of the agent. However, there exists a broad difference with respect to the pharmacokinetic behavior exhibited by the two radiolabeled preparations, as the best tumor to background ratio in the present study was achieved around 2–3 days, whereas the same was reported to be 5–7 days by Bhusari et al.⁵

The real strength of the present study lies in the systemic documentation of steps involved in the translation of ¹⁷⁷Lu[Lu]-trastuzumab prepared using medium specific activity ¹⁷⁷Lu (15–20 mCi μg⁻¹) from the laboratory to the clinic. The preliminary clinical evaluation of [¹⁷⁷Lu]Lu-trastuzumab in c-erbB2/HER2 positive breast cancer patients revealed preferential accumulation of the agent in cancerous lesions indicating the retention of targeting efficacy of trastuzumab in the radiolabeled preparation. Although the study is preliminary due to the limited number of patients included in the cohort, the concordance observed between the [¹⁸F]FDG scans and [¹⁷⁷Lu]Lu-trastuzumab scans indicates the potential of the agent towards radioimmunotheranostic applications.

Conclusion

Optimization of reaction parameters resulted in a radiochemical protocol which enabled the preparation of [¹⁷⁷Lu]Lu-trastuzumab not only in high radiolabeling yield but also in a reproducible manner. Also, [¹⁷⁷Lu]Lu-trastuzumab prepared using the optimized reaction protocol was not observed to tamper with the bio-avidity of the monoclonal antibody as the complex successfully picked up the immunohistochemically proven HER2 +ve cancerous lesions in imaging studies exhibiting concordance with [¹⁸F]FDG PET whole-body scans recorded in the same patient. In spite of the fact that the study is quite preliminary, it definitely indicates the radioimmunotheranostic potential of [¹⁷⁷Lu]Lu-trastuzumab.

Funding

Bhabha Atomic Research Centre (BARC) is a constituent unit of the Department of Atomic Energy (DAE), Government of India and all research activities carried out at the Centre are fully funded by the Government of India.

Conflicts of interest

The authors declare that they have no conflicts of interest.