Bridging the maytansine and vinca sites: Cryptophycins target ß-tubulin's T5-loop.

Cryptophycins are microtubule-targeting agents (MTAs) that belong to the most potent antimitotic compounds known to date; however, their exact molecular mechanism of action remains unclear. Here, we present the 2.2 Å resolution X-ray crystal structure of a potent cryptophycin derivative bound to the αß-tubulin heterodimer. The structure addresses conformational issues present in a previous 3.3 Å resolution cryo-electron microscopy structure of cryptophycin-52 bound to the maytansine site of ß-tubulin. It further provides atomic details on interactions of cryptophycins, which had not been described previously, including ones that are in line with structure-activity relationship studies. Interestingly, we discovered a second cryptophycin-binding site that involves the T5-loop of ß-tubulin, a critical secondary structure element involved in the exchange of the guanosine nucleotide and in the formation of longitudinal tubulin contacts in microtubules. Cryptophycins are the first natural ligands found to bind to this new "ßT5-loop site" that bridges the maytansine and vinca sites. Our results offer unique avenues to rationally design novel MTAs with the capacity to modulate T5-loop dynamics and to simultaneously engage multiple ß-tubulin binding sites.

Site-Specific Stability Evaluation of Antibody-Drug Conjugate in Serum Using a Validated Liquid Chromatography-Mass Spectrometry Method.

Antibody-drug conjugate (ADC) consists of engineered antibodies and cytotoxic drugs linked via a chemical linker, and the stability of ADC plays a crucial role in ensuring its safety and efficacy. The stability of ADC is closely related to the conjugation site; however, no method has been developed to assess the stability of different conjugation sites due to the low response of conjugated peptides. In this study, an integrated strategy was developed and validated to assess the stability of different conjugation sites on ADC in serum. Initial identification of the conjugated peptides of the model drug ado-trastuzumab emtansine (T-DM1) was achieved by the proteomic method. Subsequently, a semiquantitative method for conjugated peptides was established in liquid chromatography-hybrid linear ion trap triple quadrupole mass spectrometry (LC-QTRAP-MS/MS) based on the qualitative information. The pretreatment method of the serum sample was optimized to reduce matrix interference. The method was then validated and applied to evaluate the stability of the conjugation sites on T-DM1. The results highlighted differences in stability among the different conjugation sites on T-DM1. This is the first study to assess the stability of different conjugation sites on the ADC in serum, which will be helpful for the design and screening of ADCs in the early stages of development.

Production and characterization of a camelid single domain anti-CD22 antibody conjugated to DM1.

Antibody drug conjugates (ADCs) with twelve FDA approved drugs, known as a novel category of anti-neoplastic treatment created to merge the monoclonal antibody specificity with cytotoxicity effect of chemotherapy. However, despite many undeniable advantages, ADCs face certain problems, including insufficient internalization after binding, complex structures and large size of full antibodies especially in targeting of solid tumors. Camelid single domain antibody fragments (Nanobody®) offer solutions to this challenge by providing nanoscale size, high solubility and excellent stability, recombinant expression in bacteria, in vivo enhanced tissue penetration, and conjugation advantages. Here, an anti-human CD22 Nanobody was expressed in E.coli cells and conjugated to Mertansine (DM1) as a cytotoxic payload. The anti-CD22 Nanobody was expressed and purified by Ni-NTA resin. DM1 conjugated anti-CD22 Nanobody was generated by conjugation of SMCC-DM1 to Nanobody lysine groups. The conjugates were characterized using SDS-PAGE and Capillary electrophoresis (CE-SDS), RP-HPLC, and MALDI-TOF mass spectrometry. Additionally, flow cytometry analysis and a competition ELISA were carried out for binding evaluation. Finally, cytotoxicity of conjugates on Raji and Jurkat cell lines was assessed. The drug-to-antibody ratio (DAR) of conjugates was calculated 2.04 using UV spectrometry. SDS-PAGE, CE-SDS, HPLC, and mass spectrometry confirmed conjugation of DM1 to the Nanobody. The obtained results showed the anti-CD22 Nanobody cytotoxicity was enhanced almost 80% by conjugation with DM1. The binding of conjugates was similar to the non-conjugated anti-CD22 Nanobody in flow cytometry experiments. Concludingly, this study successfully suggest that the DM1 conjugated anti-CD22 Nanobody can be used as a novel tumor specific drug delivery system.

Synthesis and evaluation of antibody-drug conjugates with high drug-to-antibody ratio using dimaleimide-DM1 as a linker- payload.

The notable characteristics of recently emerged Antibody-Drug Conjugates (ADCs) encompass the targeting of Human Epidermal growth factor Receptor 2 (HER2) through monoclonal antibodies (mAbs) and a high ratio of drug to antibody (DAR). The achievements of Kadcyla® (T-DM1) and Enhertu® (T-Dxd) have demonstrated that HER2-targeting antibodies, such as trastuzumab, have shown to be competitive in terms of efficacy and price for development. Furthermore, with the arrival of T-Dxd and Trodelvy®, high-DAR (7-8) ADCs, which differ from the moderate DAR (3-4) ADCs that were formerly regarded as conventional, are being acknowledged for their worth. Following this trend of drug development, we endeavored to develop a high-DAR ADC using a straightforward approach involving the utilization of DM1, a highly potent substance, in combination with the widely recognized trastuzumab. To achieve a high DAR, DM1 was conjugated to reduced cysteine through the simple design and synthesis of various dimaleimide linkers with differing lengths. Using LC and MS analysis, we have demonstrated that our synthesis methodology is uncomplicated and efficacious, yielding trastuzumab-based ADCs that exhibit a remarkable degree of uniformity. These ADCs have been experimentally substantiated to exert an inhibitory effect on cancer cells in vitro, thus affirming their value as noteworthy additions to the realm of ADCs.

A novel mertansine conjugate for acid-reversible targeted drug delivery validated through the Avidin-Nucleic-Acid-NanoASsembly platform.

In targeted cancer therapy, antibody-drug-conjugates using mertansine (DM1)-based cytotoxic compounds rely on covalent bonds for drug conjugation. Consequently, the cytotoxic DM1 derivative released upon their proteolytic digestion is up to 1000-fold less potent than DM1 and lacks a bystander effect. To overcome these limitations, we developed a DM1 derivative (keto-DM1) suitable for bioconjugation through an acid-reversible hydrazone bond. Its acid-reversible hydrazone conjugate with biotin (B-Hz-DM1) was generated and tested for efficacy using the cetuximab-targeted Avidin-Nucleic-Acid-NanoASsembly (ANANAS) nanoparticle (NP) platform. NP-tethered B-Hz-DM1 is stable at neutral pH and releases its active moiety only in endosome/lysosome mimicking acidic pH. In vitro, the NP/Cetux/B-Hz-DM1 assembly showed high potency on MDA-MB231 breast cancer cells. In vivo both B-Hz-DM1 and NP/Cetux/B-Hz-DM1 reduced tumor growth. A significantly major effect was exerted by the nanoformulation, associated with an increased in situ tumor cell death. Keto-DM1 is a promising acid-reversible mertansine derivative for targeted delivery in cancer therapy.

Gatorbulin-1, a distinct cyclodepsipeptide chemotype, targets a seventh tubulin pharmacological site.

Tubulin-targeted chemotherapy has proven to be a successful and wide spectrum strategy against solid and liquid malignancies. Therefore, new ways to modulate this essential protein could lead to new antitumoral pharmacological approaches. Currently known tubulin agents bind to six distinct sites at α/ß-tubulin either promoting microtubule stabilization or depolymerization. We have discovered a seventh binding site at the tubulin intradimer interface where a novel microtubule-destabilizing cyclodepsipeptide, termed gatorbulin-1 (GB1), binds. GB1 has a unique chemotype produced by a marine cyanobacterium. We have elucidated this dual, chemical and mechanistic, novelty through multidimensional characterization, starting with bioactivity-guided natural product isolation and multinuclei NMR-based structure determination, revealing the modified pentapeptide with a functionally critical hydroxamate group; and validation by total synthesis. We have investigated the pharmacology using isogenic cancer cell screening, cellular profiling, and complementary phenotypic assays, and unveiled the underlying molecular mechanism by in vitro biochemical studies and high-resolution structural determination of the α/ß-tubulin-GB1 complex.

Identification of 5-Carboxyfluorescein as a Probe Substrate of SLC46A3 and Its Application in a Fluorescence-Based In Vitro Assay Evaluating the Interaction with SLC46A3.

The therapeutic modalities that involve the endocytosis pathway, including antibody-drug conjugates (ADCs), have recently been developed. Since the drug escape from endosomes/lysosomes is a determinant of their efficacy, it is important to optimize the escape, and the cellular evaluation system is needed. SLC46A3, a lysosomal membrane protein, has been implicated in the pharmacological efficacy of trastuzumab emtansine (T-DM1), a noncleavable ADC used for the treatment of breast cancer, and the cellular uptake efficacy of lipid-based nanoparticles. Recently, we identified the SLC46A3 function as a proton-coupled steroid conjugate and bile acid transporter, which can directly transport active catabolites of T-DM1. Thus, the rapid and convenient assay systems for evaluating the SLC46A3 function may help to facilitate ADC development and to clarify the physiological roles in endocytosis. Here, we show that SLC46A3 dC, which localizes to the plasma membrane owing to lacking a lysosomal-sorting motif, has a great ability to transport 5-carboxyfluorescein (5-CF), a fluorescent probe, in a pH-dependent manner. 5-CF uptake mediated by SLC46A3 was significantly inhibited by compounds reported to be SLC46A3 substrates/inhibitors and competitively inhibited by estrone 3-sulfate, a typical SLC46A3 substrate. The inhibition assays followed by uptake studies revealed that SG3199, a pyrrolobenzodiazepine dimer, which has been used as an ADC payload, is a substrate of SLC46A3. Accordingly, the fluorescence-based assay system for the SLC46A3 function using 5-CF can provide a valuable tool to evaluate the interaction of drugs/drug candidates with SLC46A3.

Integrated multiple analytes and semi-mechanistic population pharmacokinetic model of tusamitamab ravtansine, a DM4 anti-CEACAM5 antibody-drug conjugate.

Tusamitamab ravtansine (SAR408701) is an antibody-drug conjugate (ADC), combining a humanized monoclonal antibody (IgG1) targeting carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) and a potent cytotoxic maytansinoid derivative, DM4, inhibiting microtubule assembly. SAR408701 is currently in clinical development for the treatment of advanced solid tumors expressing CEACAM5. It is administered intravenously as a conjugated antibody with an average Drug Antibody Ratio (DAR) of 3.8. During SAR408701 clinical development, four entities were measured in plasma: conjugated antibody (SAR408701), naked antibody (NAB), DM4 and its methylated metabolite (MeDM4), both being active. Average DAR and proportions of individual DAR species were also assessed in a subset of patients. An integrated and semi-mechanistic population pharmacokinetic model describing the time-course of all entities in plasma and DAR measurements has been developed. All DAR moieties were assumed to share the same drug disposition parameters, excepted for clearance which differed for DAR0 (i.e. NAB entity). The conversion of higher DAR to lower DAR resulted in a DAR-dependent ADC deconjugation and was represented as an irreversible first-order process. Each conjugated antibody was assumed to contribute to DM4 formation. All data were fitted simultaneously and the model developed was successful in describing the pharmacokinetic profile of each entity. Such a structural model could be translated to other ADCs and gives insight of mechanistic processes governing ADC disposition. This framework will further be expanded to evaluate covariates impact on SAR408701 pharmacokinetics and its derivatives, and thus can help identifying sources of pharmacokinetic variability and potential efficacy and safety pharmacokinetic drivers.

C3 ester side chain plays a pivotal role in the antitumor activity of Maytansinoids.

Maytansinoids, the chemical derivatives of Maytansine, are commonly used as potent cytotoxic payloads in antibody-drug conjugates (ADC). Structure-activity-relationship studies had identified the C3 ester side chain as a critical element for antitumor activity of maytansinoids. The maytansinoids bearing the methyl group at C3 position with D configuration were about 100 to 400-fold less cytotoxic than their corresponding L-epimers toward various cell lines. The detailed mechanism of how chirality affects the anticancer activity remains elusive. In this study, we determined the high-resolution crystal structure of tubulin in complex with maytansinol, L-DM1-SMe and D-DM1-SMe. And we found the carbonyl oxygen atom of the ester moiety and the tail thiomethyl group at C3 side chain of L-DM1-SMe form strong intramolecular interaction with the hydroxyl at position 9 and the benzene ring, respectively, fixing the bioactive conformation and enhancing the binding affinity. Additionally, ligand-based and structure-based virtually screening methods were used to screen the commercially macrocyclic compounds library, and 15 macrocyclic structures were picketed out as putatively new maytansine-site inhibitors. Our study provides a possible strategy for the rational discovery of next-generation maytansine site inhibitors.

Targeting and extending the eukaryotic druggable genome with natural products: cytoskeletal targets of natural products.

Covering: 2014-2019We review recent progress on natural products that target cytoskeletal components, including microtubules, actin, intermediate filaments, and septins and highlight their demonstrated and potential utility in the treatment of human disease. The anticancer efficacy of microtubule targeted agents identified from plants, microbes, and marine organisms is well documented. We highlight new microtubule targeted agents currently in clinical evaluations for the treatment of drug resistant cancers and the accumulating evidence that the anticancer efficacy of these agents is not solely due to their antimitotic effects. Indeed, the effects of microtubule targeted agents on interphase microtubules are leading to their potential for more mechanistically guided use in cancers as well as neurological disease. The discussion of these agents as more targeted drugs also prompts a reevaluation of our thinking about natural products that target other components of the cytoskeleton. For instance, actin active natural products are largely considered chemical probes and non-selective toxins. However, studies utilizing these probes have uncovered aspects of actin biology that can be more specifically targeted to potentially treat cancer, neurological disorders, and infectious disease. Compounds that target intermediate filaments and septins are understudied, but their continued discovery and mechanistic evaluations have implications for numerous therapeutic indications.

First Ring-Expanded Maytansin Lactone Accessed by a New Mutasynthetic Variant.

A multiblocked mutant strain (ΔAHBA and Δasm12, asm21) of Actinosynnema pretiosum, the producer of the highly toxic maytansinoid ansamitocin, has been used for the mutasynthetic production of new proansamitocin derivatives. The use of mutant strains that are blocked in the biosynthesis of an early building block as well as in the expression of two tailoring enzymes broadens the scope of chemo-biosynthetic access to new maytansinoids. Remarkably, a ring-expanded macrolactone derived from ansamitocin was created for the first time.

Development of a Versatile and Modular Linker for Antibody-Drug Conjugates Based on Oligonucleotide Strand Pairing.

Linker design is crucial to the success of antibody-drug conjugates (ADCs). In this work, we developed a modular linker format for attaching molecular cargos to antibodies based on strand pairing between complementary oligonucleotides. We prepared antibody-oligonucleotide conjugates (AOCs) by attaching 18-mer oligonucleotides to an anti-HER2 antibody through thiol-maleimide chemistry, a method generally applicable to any immunoglobulin with interchain disulfide bridges. The hybridization of drug-bearing complementary oligonucleotides to our AOCs was rapid, stoichiometric, and sequence-specific. AOCs loaded with cytotoxic payloads were able to selectively target HER2-overexpressing cell lines such as SK-BR-3 and N87, with in vitro potencies similar to that of the marketed ADC Kadcyla (T-DM1). Our results demonstrated the potential of utilizing AOCs as a highly versatile and modular platform, where a panel of well-characterized AOCs bearing DNA, RNA, or various nucleic acid analogs, such as peptide nucleic acids, could be easily paired with any cargo of choice for a wide range of diagnostic or therapeutic applications.

Antibody drug conjugates of cleavable amino-benzoyl-maytansinoids.

ADCs based on the natural product maytansine have been successfully employed clinically. In a previous report, ADCs based on hydrophilic non-cell permeable maytansinoids was presented. The authors in this report further explore the maytansine scaffold to develop tubulin inhibitors capable of cell permeation. The research resulted in amino-benzoyl-maytansinoid payloads that were further elaborated with linkers for conjugating to antibodies. This approach was applied to MUC16 tumor targeting antibodies for ovarian cancers. A positive control ADC was evaluated alongside the amino-benzoyl-maytansinoid ADC and the efficacy observed was equivalent while the isotype control ADCs had no effect.

Peptide-Drug Conjugate-Based Nanocombination Actualizes Breast Cancer Treatment by Maytansinoid and Photothermia with the Assistance of Fluorescent and Photoacoustic Images.

To develop a highly efficient strategy against tumors, here, a nanocombination (PDC/P@HCuS) was designed and constructed to actualize chemo-phototherapy with the assistance of fluorescence (FL) and photoacoustic (PA) images. First, a type of organic-inorganic hybrid nanosystem (P@HCuS) was engineered by coupling the fluorescence-labeled amphiphilic fPEDC copolymer on the surface of hollow mesoporous copper sulfide nanoparticle (HCuS), in which HCuS was used as a photothermal and PA agent; fPEDC as a stabilizer, chromophore, and redox/pH-sensitive gatekeeper; and both of them as drug carriers. Then, a peptide-drug conjugate (cRGD-SMCC-DM1, PDC), as a molecular targeted maytansinoid, was loaded inside of P@HCuS to form PDC/P@HCuS. Next, the PDC/P@HCuS was investigated carefully with or without near-infrared (NIR) laser irradiation. In vitro, the nanocombination exhibited stimuli-responsive drug release, obvious cellular uptake, strong cytotoxicity to tumor cells, significant impact on cell cycle, and cytoskeleton and cellular proteomics as well as evident permeability into the tumor sphere, most of which could be boosted by NIR laser irradiation. In vivo, the nanocombinaiton exerted good FL/PA imaging features and photothermal efficiency, achieved the best antitumor efficacy in the presence of NIR laser irradiation, and showed excellent biosafety. Together, it demonstrated that the PDC/P@HCuS, representing a chemo-phototherapy based on a nanocombination associated with peptide-drug conjugate, could achieve the highly efficient antitumor effect.

Chemoselective and Site-Selective Lysine-Directed Lysine Modification Enables Single-Site Labeling of Native Proteins.

The necessity for precision labeling of proteins emerged during the efforts to understand and regulate their structure and function. It demands selective attachment of tags such as affinity probes, fluorophores, and potent cytotoxins. Here, we report a method that enables single-site labeling of a high-frequency Lys residue in the native proteins. At first, the enabling reagent forms stabilized imines with multiple solvent-accessible Lys residues chemoselectively. These linchpins create the opportunity to regulate the position of a second Lys-selective electrophile connected by a spacer. Consequently, it enables the irreversible single-site labeling of a Lys residue independent of its place in the reactivity order. The user-friendly protocol involves a series of steps to deconvolute and address chemoselectivity, site-selectivity, and modularity. Also, it delivers ordered immobilization and analytically pure probe-tagged proteins. Besides, the methodology provides access to antibody-drug conjugate (ADC), which exhibits highly selective anti-proliferative activity towards HER-2 expressing SKBR-3 breast cancer cells.

The novel CD19-targeting antibody-drug conjugate huB4-DGN462 shows improved anti-tumor activity compared to SAR3419 in CD19-positive lymphoma and leukemia models.

Antibody-drug conjugates (ADC) are a novel way to deliver potent cytotoxic compounds to cells expressing a specific antigen. Four ADC targeting CD19, including SAR3419 (coltuximab ravtansine), have entered clinical development. Here, we present huB4-DGN462, a novel ADC based on the SAR3419 anti-CD19 antibody linked via sulfo-SPDB to the potent DNA-alkylating agent DGN462. huB4-DGN462 had improved in vitro anti-proliferative and cytotoxic activity compared to SAR3419 across multiple B-cell lymphoma and human acute lymphoblastic leukemia cell lines. In vivo experiments using lymphoma xenografts models confirmed the in vitro data. The response of B-cell lymphoma lines to huB4-DGN462 was not correlated with CD19 expression, the presence of BCL2 or MYC translocations, TP53 inactivation or lymphoma histology. In conclusion, huB4-DGN462 is an attractive candidate for clinical investigation in patients with B-cell malignancies.

Folate Receptor α-Targeted 89Zr-M9346A Immuno-PET for Image-Guided Intervention with Mirvetuximab Soravtansine in Triple-Negative Breast Cancer.

Folate receptor α (FRα) is a well-studied tumor biomarker highly expressed in many epithelial tumors such as breast, ovarian, and lung cancers. Mirvetuximab soravtansine (IMGN853) is the antibody-drug conjugate of FRα-binding humanized monoclonal antibody M9346A and cytotoxic maytansinoid drug DM4. IMGN853 is currently being evaluated in multiple clinical trials, in which the immunohistochemical evaluation of an archival tumor or biopsy specimen is used for patient screening. However, limited tissue collection may lead to inaccurate diagnosis due to tumor heterogeneity. Herein, we developed a zirconium-89 (89Zr)-radiolabeled M9346A (89Zr-M9346A) as an immuno-positron emission tomography (immuno-PET) radiotracer to evaluate FRα expression in triple-negative breast cancer (TNBC) patients, providing a novel means to guide intervention with therapeutic IMGN853. In this study, we verified the binding specificity and immunoreactivity of 89Zr-M9346A by in vitro studies in FRαhigh cells (HeLa) and FRαlow cells (OVCAR-3). In vivo PET/computed tomography (PET/CT) imaging in HeLa xenografts and TNBC patient-derived xenograft (PDX) mouse models with various levels of FRα expression demonstrated its targeting specificity and sensitivity. Following PET imaging, the treatment efficiencies of IMGN853, pemetrexed, IMGN853 + pemetrexed, paclitaxel, and saline were assessed in FRαhigh and FRαlow TNBC PDX models. The correlation between 89Zr-M9346A tumor uptake and treatment response using IMGN853 in FRαhigh TNBC PDX model suggested the potential of 89Zr-M9346A PET as a noninvasive tool to prescreen patients based on the in vivo PET imaging for IMGN853-targeted treatment.

Design, synthesis and evaluation of anti-CD38 antibody drug conjugate based on Daratumumab and maytansinoid.

Daratumumab, an FDA approved antibody drug, displays specific targeting ability to abnormal white blood cells overexpressing CD38 and provides efficacious therapy for multiple myeloma. Here, in order to achieve enhanced remission of multiple myeloma, we designed Dara-DM4, antibody drug conjugates (ADCs) by conjugating Daratumumab and DM4 via a disulfide linker. Dara-DM4 showed significantly higher cellular uptake and inhibitory efficacy on MM1S cells that overexpressing CD38 with an IC50 of 0.88 µg/mL post 72 hr treatment. These results support a promising ADCs strategy for multiple myeloma treatment.

Bioengineered Macrophages Can Responsively Transform into Nanovesicles To Target Lung Metastasis.

Specific drug delivery to metastatic tumors remains a great challenge for antimetastasis therapy. We herein report a bioengineered macrophage-based delivery system (LD-MDS) that can be preferentially delivered to lung metastases and intelligently transformed into nanovesicles and secondary nanovesicles for antimetastasis therapy. LD-MDS was prepared by anchoring a legumain-specific propeptide of melittin (legM) and cytotoxic soravtansine (DM4) prodrug onto the membrane of living macrophages. LD-MDS is responsively activated by legumain protease and converted into DM4-loaded exosome-like nanovesicles (DENs), facilitating efficient internalization by metastatic 4T1 cancer cells and considerable cell death. Afterward, the damaged 4T1 cells can release secondary nanovesicles and free drug molecules to destroy neighboring cancer cells. In vivo, LD-MDS displays superior targeting efficiency for lung metastatic lesions with diameters less than 100 µm and remarkably inhibits lung metastasis. This study provides a new opportunity to explore endogenous macrophages as living drug delivery vehicles with controlled drug release to target metastatic lung tumors.

Positron-Emission Tomography of HER2-Positive Breast Cancer Xenografts in Mice with 89Zr-Labeled Trastuzumab-DM1: A Comparison with 89Zr-Labeled Trastuzumab.

Our aim was to synthesize 89Zr-labeled trastuzumab-emtansine (89Zr-DFO-T-DM1) to probe the delivery of trastuzumab-emtansine (T-DM1) to HER2-positive breast cancer (BC) by positron emission tomography (PET). We further aimed to compare the tumor and normal tissue uptake of 89Zr-DFO-T-DM1 with 89Zr-DFO-trastuzumab. T-DM1 was modified with 3.0 ± 0.2 desferrioxamine (DFO) chelators for complexing 89Zr by reaction with a 14-fold molar excess of p-NCS-Bz-DFO. The number of DFO chelators per T-DM1 molecule was quantified spectrophotometrically at 430 nm after the reaction with FeCl3. SDS-PAGE and SE-HPLC demonstrated a pure and homogeneous immunoconjugate. DFO-T-DM1 and DFO-trastuzumab were labeled to high efficiency (>97%) with 89Zr at a specific activity of 0.55 MBq/µg in a 2 M Na2CO3/0.5 M HEPES buffer, pH 7.0, at RT for 60-90 min. The labeling efficiency was measured by instant thin layer-silica gel chromatography (ITLC-SG) and SE-HPLC. HER2 immunoreactivity was measured in a saturation binding assay using SK-BR-3 human BC cells. 89Zr-DFO-T-DM1 exhibited high affinity HER2 binding ( Kd = 3.7 ± 0.4 nM) that was not significantly different than 89Zr-DFO-trastuzumab (4.4 ± 0.5 nM; P = 0.06). The optimal time for tumor imaging with 89Zr-DFO-T-DM1 was 96 h post-injection in NOD-scid mice with s.c. HER2 overexpressing (HER2 3+) BT-474 human BC xenografts. Tumor uptake was dependent on the level of HER2 expression in mice with s.c. BT-474 (HER2 3+), MDA-MB-231/H2N (HER2 2+), MDA-MB-231 (HER2 0-1+), or MDA-MB-468 (HER2 0) human BC xenografts injected with 89Zr-DFO-T-DM1 (10 µg, 5.2 MBq). All tumors were visualized by microPET/CT, but the tumor intensity was greatest for BT-474 and MDA-MB-231/H2N xenografts. The tumor uptake of 89Zr-DFO-T-DM1 was 4.1-fold significantly higher than 89Zr-DFO-trastuzumab in mice with s.c. BT-474 (HER2 3+) xenografts (43.5 ± 4.3%ID/g vs 10.6 ± 5.4%ID/g, respectively; P < 0.001). Tumor uptake of 89Zr-DFO-T-DM1 in MDA-MB-231/H2N xenografts (HER2 2+) was 3.7-fold significantly higher than 89Zr-DFO-trastuzumab (10.1 ± 3.6%ID/g vs 2.7 ± 0.5%ID/g; P < 0.001). The higher tumor uptake of 89Zr-DFO-T-DM1 compared to 89Zr-DFO-trastuzumab was not due to a higher HER2 binding affinity or to differences in the residence time in the blood or tumor size. We conclude that 89Zr-DFO-T-DM1 is a useful probe to assess the delivery of T-DM1 to HER2-positive BC. PET with 89Zr-DFO-trastuzumab has been studied clinically to predict response to T-DM1, but our results suggest that 89Zr-DFO-T-DM1 may be more accurate due to the differences in the tumor uptake observed in the preclinical BC xenograft mouse models.