Tumor Targeting Using Affibody Molecules: Interplay of Affinity, Target Expression Level, and Binding Site Composition

Affibody molecules are a class of affinity proteins. Their small size (7 kDa) in combination with the high (subnanomolar) affinity for a number of cancer-associated molecular targets makes them suitable for molecular imaging. Earlier studies demonstrated that the selection of radionuclide and chelator may substantially influence the tumor-targeting properties of affibody molecules. Moreover, the placement of chelators for labeling of affibody molecules with (99m)Tc at different positions in affibody molecules influenced both blood clearance rate and uptake in healthy tissues. This introduces an opportunity to improve the contrast of affibody-mediated imaging. In this comparative study, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the synthetic affibody molecule Z(HER2:S1) at three different positions: DOTA-A1-Z(HER2:S1) (N-terminus), DOTA-K58-Z(HER2:S1) (C-terminus), and DOTA-K50-Z(HER2:S1) (middle of helix 3). The affinity for HER2 differed slightly among the variants and the K(D) values were determined to be 133 pM, 107 pM and 94 pM for DOTA-A1-Z(HER2:S1), DOTA-K50-Z(HER2:S1), and DOTA-K58-Z(HER2:S1), respectively. Z(HER2:S1)-K50-DOTA showed a slightly lower melting point (57 °C) compared to DOTA-A1-Z(HER2:S1) (64 °C) and DOTA-K58-Z(HER2:S1) (62 °C), but all variants showed good refolding properties after heat treatment. All conjugates were successfully labeled with (111)In resulting in a radiochemical yield of 99% with preserved binding capacity. In vitro specificity studies using SKOV-3 and LS174T cell lines showed that the binding of the radiolabeled compounds was HER2 receptor-mediated, which also was verified in vivo using BALB/C nu/nu mice with LS174T and Ramos lymphoma xenografts. The three conjugates all showed specific uptake in LS174T xenografts in nude mice, where DOTA-A1-Z(HER2:S1)and DOTA-K58-Z(HER2:S1) showed the highest uptake. Overall, DOTA-K58-Z(HER2:S1) provided the highest tumor-to-blood ratio, which is important for a high-contrast imaging. In conclusion, the positioning of the DOTA chelator influences the cellular processing and the biodistribution pattern of radiolabeled affibody molecules, creating preconditions for imaging optimization.

Overexpression of epidermal growth factor receptor (EGFR) is a prognostic and predictive biomarker in a number of malignant tumours. Radionuclide molecular imaging of EGFR expression in cancer could influence patient management. However, EGFR expression in normal tissues might complicate in vivo imaging. The aim of this study was to evaluate if optimization of the injected protein dose might improve imaging of EGFR expression in tumours using a novel EGFR-targeting protein, the DOTA-Z(EGFR:2377) Affibody molecule. An anti-EGFR Affibody molecule, Z(EGFR:2377), was labelled with (111)In via the DOTA chelator site-specifically conjugated to a C-terminal cysteine. The affinity of DOTA-Z(EGFR:2377) for murine and human EGFR was measured by surface plasmon resonance. The cellular processing of (111)In-DOTA-Z(EGFR:2377) was evaluated in vitro. The biodistribution of radiolabelled Affibody molecules injected in a broad range of injected Affibody protein doses was evaluated in mice bearing EGFR-expressing A431 xenografts. Site-specific coupling of DOTA provided a uniform conjugate possessing equal affinity for human and murine EGFR. The internalization of (111)In-DOTA-Z(EGFR:2377) by A431 cells was slow. In vivo, the conjugate accumulated specifically in xenografts and in EGFR-expressing tissues. The curve representing the dependence of tumour uptake on the injected Affibody protein dose was bell-shaped. The highest specific radioactivity (lowest injected protein dose) provided a suboptimal tumour-to-blood ratio. The results of the biodistribution study were confirmed by gamma-camera imaging. The (111)In-DOTA-Z(EGFR:2377) Affibody molecule is a promising tracer for radionuclide molecular imaging of EGFR expression in malignant tumours. Careful optimization of protein dose is required for high-contrast imaging of EGFR expression in vivo.

Affibody molecules are low molecular weight proteins (7 kDa), which can be selected to bind to tumour-associated target proteins with subnanomolar affinity. Because of rapid tumour localisation and clearance from nonspecific compartments, Affibody molecules are promising tracers for molecular imaging. Earlier, (99m)Tc-labelled Affibody molecules demonstrated specific targeting of tumour xenografts. However, the biodistribution was suboptimal either because of hepatobiliary excretion or high renal uptake of the radioactivity. The goal of this study was to optimise the biodistribution of Affibody molecules by chelator engineering. Anti-HER2 Z(HER2:342) Affibody molecules, carrying the mercaptoacetyl-glutamyl-seryl-glutamyl (maESE), mercaptoacetyl-glutamyl-glutamyl-seryl (maEES) and mercaptoacetyl-seryl-glutamyl-glutamyl (maSEE) chelators, were prepared by peptide synthesis and labelled with (99m)Tc. The tumour-targeting capacity of these conjugates was compared with each other and with the best previously available conjugate, (99m)Tc-maEEE-Z(HER2:342,) in nude mice bearing SKOV-3 xenografts. The tumour-targeting capacity of the most promising conjugate, (99m)Tc-maESE-Z(HER2:342,) was compared with radioiodinated Z(HER2:342). All novel conjugates demonstrated successful tumour targeting and a low degree of hepatobiliary excretion. The renal uptakes of serine-containing conjugates, 33 +/- 5, 68 +/- 21 and 71 +/- 10%IA/g, for(99m)Tc-maESE-Z(HER2:342), (99m)Tc-maEES-Z(HER2:342) and (99m)Tc-maSEE-Z(HER2:342), respectively, were significantly reduced in comparison with (99m)Tc-maEEE-Z(HER2:342) (102 +/- 13%IA/g). For (99m)Tc-maESE-Z(HER2:342), a tumour uptake of 9.6 +/- 1.8%IA/g and a tumour-to-blood ratio of 58 +/- 6 were reached at 4 h p.i. A combination of serine and glutamic acid residues in the chelator sequence confers increased renal excretion and relatively low renal uptake of (99m)Tc-labelled Affibody molecules. In combination with preserved targeting capacity, this improved imaging of targets in abdominal area.

Design of an Optimized Scaffold for Affibody Molecules

Because of their better penetration, smaller targeting proteins may be superior to antibodies for radioimmunotherapy of solid tumors. Therefore, Affibody molecules (6.5 kDa) have a potential for being suitable as targeted moiety for radiolabeled therapeutic proteins. Previous studies have demonstrated that a fusion of an Affibody molecule with an albumin-binding domain (ABD) provides a strong noncovalent binding to albumin in vivo. This strong noncovalent binding can be used for reduction of the renal uptake of the Affibody molecule while maintaining a size smaller than that of an antibody, which is important when using residualizing radionuclide labels conjugated to Affibody molecules. The goal of this study was to design and evaluate a new targeting Affibody-ABD fusion protein with improved biodistribution properties for radionuclide therapy. A novel Affibody-based construct, ZHER2:2891-ABD035-DOTA (ABY-027), was created by fusion of the reengineered HER2-binding Affibody molecule ZHER2:2891 to the N terminus of the high-affinity ABD035, and a maleimido-derivative of DOTA was conjugated at the C terminus of the construct. Binding and processing of (177)Lu-ABY-027 by HER2-expressing cells were evaluated in vitro. Targeting of HER2-expressing SKOV-3 xenografts was evaluated in BALB/C nu/nu mice and compared with targeting of previously reported ABD-(ZHER2:342)2. The binding affinity (dissociation constant) of ABY-027 to HER2 (74 pM) was the same as for the parental ZHER2:2891 (76 pM). ABY-027 was stably labeled with (177)Lu and (111)In with preserved specific binding to HER2-expressing cells in vitro. In vivo receptor saturation experiments demonstrated that targeting of SKOV-3 xenografts in BALB/C nu/nu mice was HER2-specific. (177)Lu-ABY-027 demonstrated substantially (2- to 3-fold) lower renal and hepatic uptake than previously assessed HER2-specific Affibody-based albumin-binding agents. Tumor uptake of radiolabeled ABY-027 at 48 h after injection was 2-fold higher than that for previously reported ABD-(ZHER2:342)2. An optimized molecular design of an ABD fusion protein resulted in an Affibody molecule construct with better properties for therapy. Fully preserved in vivo targeting of the fusion protein was shown in xenografted mice. Site-specific coupling of DOTA provides a uniform conjugate and creates the potential for labeling with a broad range of therapeutic radionuclides. The biodistribution of (177)Lu-ABY-027 in a murine model suggests it is more suitable for therapy than alternative approaches.

Simple SummaryAffibody molecules are artificial proteins that can recognize cancer-related molecular abnormalities in the living body. Clinical studies demonstrated that Affibody molecules can be successfully used for radionuclide diagnostics. Targeted radionuclide therapy selectively delivers cytotoxic radionuclides to malignant tumors, thus sparing normal tissues. For radionuclide therapy, Affibody molecules were re-engineered to decrease accumulation in the kidneys. This study has demonstrated that radionuclide therapy using re-engineered Affibody molecules increases the survival of immunodeficient mice bearing human tumors. The therapy was more efficient than the treatment with a monoclonal antibody, which is currently used in clinical practice. The best results were obtained when both the antibody and radiolabeled Affibody molecules were used simultaneously. This work provides a preclinical rationale for a potentially more efficient treatment in HER2-positive cancers.ABY-027 is a scaffold-protein-based cancer-targeting agent. ABY-027 includes the second-generation Affibody molecule ZHER2:2891, which binds to human epidermal growth factor receptor type 2 (HER2). An engineered albumin-binding domain is fused to ZHER2:2891 to reduce renal uptake and increase bioavailability. The agent can be site-specifically labeled with a beta-emitting radionuclide 177Lu using a DOTA chelator. The goals of this study were to test the hypotheses that a targeted radionuclide therapy using [177Lu]Lu-ABY-027 could extend the survival of mice with HER2-expressing human xenografts and that co-treatment with [177Lu]Lu-ABY-027 and the HER2-targeting antibody trastuzumab could enhance this effect. Balb/C nu/nu mice bearing HER2-expressing SKOV-3 xenografts were used as in vivo models. A pre-injection of trastuzumab did not reduce the uptake of [177Lu]Lu-ABY-027 in tumors. Mice were treated with [177Lu]Lu-ABY-027 or trastuzumab as monotherapies and a combination of these therapies. Mice treated with vehicle or unlabeled ABY-027 were used as controls. Targeted monotherapy using [177Lu]Lu-ABY-027 improved the survival of mice and was more efficient than trastuzumab monotherapy. A combination of therapies utilizing [177Lu]Lu-ABY-027 and trastuzumab improved the treatment outcome in comparison with monotherapies using these agents. In conclusion, [177Lu]Lu-ABY-027 alone or in combination with trastuzumab could be a new potential agent for the treatment of HER2-expressing tumors.

Background: Human epidermal growth factor receptor type 2 (HER2) is abundant in a wide variety of tumors and associated with the poor prognosis. Radiolabeled affibodies are potential candidates for detecting HER2-positive lesions. However, laborious multiple-step synthetic procedure and high abdomen background may hinder the widespread use. Herein, cysteinylated ZHER2:342 modified with a new hydrophilic linker (denoted as MZHER2:342) was designed and labeled using 18FAl-NOTA strategies. The biologic efficacy of the novel tracer and its feasibilities for in vivo monitoring HER2 levels were also investigated in xenograft models with different HER2 expressions.Method: MZHER2:342 was conjugated with MAL-NOTA under standard reaction conditions. The affibody molecule was then radiolabeled with 18FAl complex. The binding specificity of the tracer, 18FAl-NOTA-MAL-MZHER2:342, with HER2 was primarily characterized via in vitro studies. MicroPET imaging were performed in nude mice bearing tumors (SKOV-3, JIMT-1 and MCF-7) after injection. The HER2 levels of xenografts were determined using Western blotting analysis.Results:18FAl-NOTA-MAL-MZHER2:342 can be efficiently produced within 30 min with a non-decaycorrected yield of about 10% and a radiochemical purity of more than 95%. In vitro experiments revealed that the modified affibody retained the specific affinity to HER2. PET imaging showed that SKOV-3 and JIMT-1 xenografts were clearly visualized with excellent contrast and low abdomen backgrounds. On the contrary, the signals of MCF-7 tumor were difficult to visualize. The ROI values ranged from16.54±2.69% ID/g for SKOV-3 to 8.42±1.20 %ID/g for JIMT-1 tumors at 1h postinjection respectively. Poor uptake was observed from MCF-7 tumors with 1.71±0.34% ID/g at the same time point. Besides, a significant linear correlation between % ID/g values and relative HER2 expression levels was also found.Conclusions:18FAl-NOTA-MAL-MZHER2:342 is a promising tracer for in vivo detecting HER2 status with the advantages of facile synthesis and favorable pharmacokinetics. It may be useful in differential diagnosis, molecularly targeted therapy and prognosis of the cancers.

Overexpression of the human epidermal growth factor receptor type 2 (HER2) in breast carcinomas predicts response to trastuzumab therapy. Affibody molecules based on a nonimmunoglobulin scaffold have demonstrated a high potential for in vivo molecular imaging of HER2-expressing tumors. The reengineering of the molecular scaffold has led to a second generation of optimized Affibody molecules that have a surface distinctly different from the parental protein domain from staphylococcal protein A. Compared with the parental molecule, the new tracer showed a further increased melting point, stability, and overall hydrophilicity and was more amenable to chemical peptide synthesis. The goal of this study was to assess the potential effects of this extensive reengineering on HER2 targeting, using ABY-025, a DOTA-conjugated variant of the novel tracer. (111)In-ABY-025 was compared with previously evaluated parent HER2-binding Affibody tracers in vitro and in vivo. The in vivo behavior was further evaluated in mice bearing SKOV-3 xenografts, rats, and cynomolgus macaques (Macaca fascicularis). (111)In-ABY-025 bound specifically to HER2 in vitro and in vivo. Direct comparison with the previous generation of HER2-binding tracers showed that ABY-025 retained excellent targeting properties. Rapid blood clearance was shown in mice, rats, and macaques. A highly specific tumor uptake of 16.7 +/- 2.5 percentage injected activity per gram of tissue was seen at 4 h after injection. The tumor-to-blood ratio was 6.3 at 0.5 h and 88 at 4 h and increased up to 3 d after injection. gamma-camera imaging of tumors was already possible at 0.5 h after injection. Furthermore, the repeated intravenous administration of ABY-025 did not induce antibody formation in rats. The biodistribution of (111)In-ABY-025 was in remarkably good agreement with the parent tracers, despite profound reengineering of the nonbinding surface. The molecule displayed rapid blood clearance in all species investigated and excellent targeting capacity in tumor-bearing mice, leading to high tumor-to-organ-ratios and high-contrast imaging shortly after injection.

Coexpression of several ErbB receptors has been found in many cancers and has been linked with increased aggressiveness of tumors and a worse patient prognosis. This makes the simultaneous targeting of two surface receptors by using bispecific constructs an increasingly appreciated strategy. Here, we have generated six such bispecific targeting proteins, each comprising two monomeric affibody molecules with specific binding to either of the two human epidermal growth factor receptors, EGFR and HER2, respectively. The bispecific constructs were designed with (i) alternative positioning (N- or C-terminal) of the different affibody molecules, (ii) two alternative peptide linkers (Gly(4)Ser)(3) or (Ser(4)Gly)(3), and (iii) affibody molecules with different affinity (nanomolar or picomolar) for HER2. Using both Biacore technology and cell binding assays, it was demonstrated that all six constructs could bind simultaneously to both their target proteins. N-terminal positioning of the inherent monomeric affibody molecules was favorable to promote the binding to the respective target. Interestingly, bispecific constructs containing the novel (Ser(4)Gly)(3) linker displayed a higher affinity in cell binding, as compared to constructs containing the more conventional linker, (Gly(4)Ser)(3). It could further be concluded that bispecific constructs (but not the monomeric affibody molecules) induced dimer formation and phosphorylation of EGFR in SKBR3 cells, which express fairly high levels of both receptors. It was also investigated whether the bispecific binding would influence cell growth or sensitize cells for ionizing radiation, but no such effects were observed.

Affibody molecules, small (7 kDa) scaffold proteins, are a promising class of probes for radionuclide molecular imaging. Radiolabeling of Affibody molecules with the positron-emitting nuclide 68Ga would permit the use of positron emission tomography (PET), providing better resolution, sensitivity, and quantification accuracy than single-photon emission computed tomography (SPECT). The synthetic anti-HER2 ZHER2:S1 Affibody molecule was conjugated with DOTA at the N-terminus, in the middle of helix 3, or at the C-terminus. The biodistribution of 68Ga- and 111In-labeled Affibody molecules was directly compared in NMRI nu/nu mice bearing SKOV3 xenografts. The position of the chelator strongly influenced the biodistribution of the tracers, and the influence was more pronounced for 68Ga-labeled Affibody molecules than for the 111In-labeled counterparts. The best 68Ga-labeled variant was 68Ga-[DOTA-A1]-ZHER2:S1, which provided a tumor uptake of 13 ± 1 %ID/g and a tumor to blood ratio of 39 ± 12 at 2 hours after injection. 111In-[DOTA-A1]-ZHER2:S1 and 111In-[DOTA-K58]-ZHER2:S1 were equally good at this time point, providing a tumor uptake of 15 to 16 %ID/g and a tumor to blood ratio in the range of 60 to 80. In conclusion, the selection of the best position for a chelator in Affibody molecules can be used for optimization of their imaging properties. This may be important for the development of Affibody-based and other protein-based imaging probes.

Objective To investigate the expression of microRNA (miRNA, miR)-126-3p in breast carcinomas with four different immunohistochemical profiling and their relevance to clinicopathologic features. Methods The expression of miR-126-3p was detected by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) in 67 paired frozen breast carcinomas tissues and further detected by TMA-ISH in 140 cases of formalin fixed paraffin-embedded (FFPE) breast carcinomas tissues. Results MiR-126-3p expression level was lower in carcinoma tissues than that in paired para-cancerous tissues, and lowest in triple-negative breast cancer (TNBC) immune phenotype without significant difference using FQ-PCR method. The results of TMA-ISH showed that miR-126-3p was mainly expressed in stroma of tumor tissue, and its expression level was higher in tumor tissue than that in breast fibroadenomas (χ2=21.48, P<0.01). Triple-negative breast cancers in stroma showed the lowest miR-126-3p expression level (high expression rate of 60%), and human epidermal growth factor receptor-2 (Her-2) over-expressed group showed the highest expression level (high expression rate of 91.3%) within four subtypes, which had the significant differences (χ2=6.26; P<0.05). Furthermore, strong miR-126-3p expression was significantly associated with Her-2 expression (χ2=5.73, P<0.05). Conclusion There was a difference in the miR-126-3p expression in breast tumor between different immune phenotypes, which may be used as a potential individual diagnostic and drug target for breast cancer patients. Key words: Breast carcinoma; MicroRNA-126-3p; Human epidermal growth factor receptor-2 over-expression

The human epidermal growth factor receptor 2 (HER2) is a clinically validated target for cancer therapy, and targeted therapies are often used in regimens for patients with a high HER2 expression level. Despite the success of current drugs, a number of patients succumb to their disease, which motivates development of novel drugs with other modes of action. We have previously shown that an albumin binding domain-derived affinity protein with specific affinity for HER2, ADAPT6, can be used to deliver the highly cytotoxic protein domain PE25, a derivative of Pseudomonas exotoxin A, to HER2 overexpressing malignant cells, leading to potent and specific cell killing. In this study we expanded the investigation for an optimal targeting domain and constructed two fusion toxins where a HER2-binding affibody molecule, ZHER2:2891, or the dual-HER2-binding hybrid ZHER2:2891-ADAPT6 were used for cancer cell targeting. We found that both targeting domains conferred strong binding to HER2; both to the purified extracellular domain and to the HER2 overexpressing cell line SKOV3. This resulted in fusion toxins with high cytotoxic potency toward cell lines with high expression levels of HER2, with EC50 values between 10 and 100 pM. For extension of the plasma half-life, an albumin binding domain was also included. Intravenous injection of the fusion toxins into mice showed a profound influence of the targeting domain on biodistribution. Compared to previous results, with ADAPT6 as targeting domain, ZHER2:2891 gave rise to further extension of the plasma half-life and also shifted the clearance route of the fusion toxin from the liver to the kidneys. Collectively, the results show that the targeting domain has a major impact on uptake of PE25-based fusion toxins in different organs. The results also show that PE25-based fusion toxins with high affinity to HER2 do not necessarily increase the cytotoxicity beyond a certain point in affinity. In conclusion, ZHER2:2891 has the most favorable characteristics as targeting domain for PE25.

Introduction: U3-1402 is a novel, investigational ADC directed against HER3, which is widely expressed in breast cancer. Safety and preliminary antitumor activity of U3-1402 was previously reported in this ongoing, phase 1/2 clinical trial (NCT02980341/JapicCTI-163401). Here we report updated safety data from the dose escalation/dose finding (DE/DF) and phase 2 dose expansion parts, and we present for the first time efficacy data for patients in the expansion part with HR+/HER2− MBC with high or low levels of HER3 expression. Methods: In DE/DF, U3-1402 was administered intravenously (IV) Q2W or Q3W at doses ranging from 1.6 to 8.0 mg/kg. Patients had HER3-expressing advanced/unresectable disease refractory/intolerant to standard treatment or for which no standard treatment was available. In the expansion part, U3-1402 was administered IV Q3W to patients with HER3-high (4.8 or 6.4 mg/kg) or HER3-low (6.4 mg/kg) HR+/HER2− MBC or with HER3-high triple-negative breast cancer (TNBC; 6.4 mg/kg). Primary objectives included safety and efficacy of U3-1402; secondary objectives included correlative biomarker and pharmacokinetics analyses. Archival or pretreatment tissue obtained in the 6 months prior to U3-1402 treatment was analyzed by IHC for HER3 expression. Results: As of March 2020, all 4 expansion cohorts had completed enrollment. In the DE/DF and expansion parts combined, 172 patients received ≥1 dose of U3-1402 (114 [66%] had HR+/HER2− MBC). Median age was 56 y (range, 30-83 y); 123 (72%) and 49 (28%) patients had an ECOG PS of 0 or 1, respectively. Patients received a median of 6 (range, 1-14) prior systemic therapies. Median treatment duration of U3-1402 was 5 mo (range, 1-30 mo). 101 patients (59%) discontinued because of progressive disease, and 15 (9%) discontinued because of an AE. The most common grade ≥3 treatment-emergent AEs reported were decreased neutrophil count (43 patients [25%]), decreased platelet count (39 patients [23%]), decreased white blood cell count (28 patients [16%]), and anemia (31 patients [18%]). Nine patients (5%) experienced treatment-related interstitial lung disease according to central adjudication, including one grade 5 event. At data cutoff, 85 patients in the HR+/HER2− expansion part were evaluable for efficacy by blinded independent central review. Among 33 patients with HR+/HER2− HER3-high MBC treated with 4.8 mg/kg, 10 (30%) had a response (all partial responses [PRs]), with a clinical benefit rate (CBR; complete response + PR + stable disease for ≥6 mo) of 45% (15/33). Among 31 patients with HER3-high MBC treated with 6.4 mg/kg, 3 (10%) had a response (all PR) with a CBR of 16% (5/31). Among 21 patients with HER3-low MBC treated with 6.4 mg/kg, 6 (29%) had a response (all PRs) with a CBR of 33% (7/21). TNBC data are immature at the time of abstract data cutoff; efficacy by HR status will be provided in the presentation. Membrane HER3 expression levels were dynamic; they varied between archival and pretreatment biopsies and decreased on treatment with U3-1402. Across the doses evaluated, the U3-1402 AUC and Cmax of the total antibody, intact ADC, and released payload were approximately dose proportional, with minimal accumulation after multiple doses; the released payload Cmax was highest in cycle 1 and decreased in later cycles. Conclusions: In this heavily pretreated MBC population, U3-1402 demonstrated clinically meaningful antitumor activity in HR+/HER2− MBC with high or low levels of HER3 expression. The safety profile of U3-1402 was consistent with that previously reported, with 9% of patients discontinuing because of AEs. Clinical data pertaining to patients with TNBC and correlative analyses (including association of HER3 expression with response) will be presented. Citation Format: Ian Krop, Kan Yonemori, Shunji Takahashi, Kenichi Inoue, Takahiro Nakayama, Hiroji Iwata, Tatsuya Toyama, Yutaka Yamamoto, Masato Takahashi, Akihiko Osaki, Shigehira Saji, Yasuaki Sagara, Joyce O’Shaughnessy, Tiffany Traina, Shoichi Ohwada, Zhenhao Qi, Yang Qiu, Hiroshi Onuma, Om Sharma, Sabeen Mekan, Norikazu Masuda. Safety and efficacy results from the phase 1/2 study of U3-1402, a human epidermal growth factor receptor 3 (HER3)-directed antibody drug conjugate (ADC), in patients with HER3-expressing metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD1-09.

BackgroundRadionuclide molecular imaging can be used to visualize the expression levels of molecular targets. Affibody molecules, small and high affinity non-immunoglobulin scaffold-based proteins, have demonstrated promising properties as targeting vectors for radionuclide tumour imaging of different molecular targets. B7-H3 (CD276), an immune checkpoint protein belonging to the B7 family, is overexpressed in different types of human malignancies. Visualization of overexpression of B7-H3 in malignancies enables stratification of patients for personalized therapies. Affinity maturation of anti-B7-H3 Affibody molecules as an approach to improve the binding affinity and targeting properties was recently investigated. In this study, we tested the hypothesis that a dimeric format may be an alternative option to increase the apparent affinity of Affibody molecules to B7-H3 and accordingly improve imaging contrast.ResultsTwo dimeric variants of anti-B7-H3 Affibody molecules were produced (designated ZAC12*-ZAC12*-GGGC and ZAC12*-ZTaq_3-GGGC). Both variants were labelled with Tc-99m (99mTc) and demonstrated specific binding to B7-H3-expressing cells in vitro. [99mTc]Tc-ZAC12*-ZAC12*-GGGC showed subnanomolar affinity (KD1=0.28 ± 0.10 nM, weight = 68%), which was 7.6-fold higher than for [99mTc]Tc-ZAC12*-ZTaq_3-GGGC (KD=2.1 ± 0.9 nM). Head-to-head biodistribution of both dimeric variants of Affibody molecules compared with monomeric affinity matured SYNT-179 (all labelled with 99mTc) in mice bearing B7-H3-expressing SKOV-3 xenografts demonstrates that both dimers have lower tumour uptake and lower tumour-to-organ ratios compared to the SYNT-179 Affibody molecule.ConclusionThe improved functional affinity by dimerization does not compensate the disadvantage of increased molecular size for imaging purposes.

3069 Background: Over-expression the human epidermal growth factor receptor-2 (HER2) is seen in 20% of breast cancers; this is an adverse prognostic factor and used to guide therapy selection. At present HER2 expression can only be determined using biopsy material using immunohistochemistry or fluorescence in situ hybridisation. Heterogeneous expression of HER2 is now being recognised as a cause of treatment resistance but is difficult to characterise. A non-invasive method for determining HER2 expression could have several advantages and help select appropriate therapy for patients. GE-226 is a novel radiolabelled GE-Affibody radioligand which binds to the HER2 receptor with high affinity at a different epitope than trastuzumab. Methods: Patients with locally advanced or metastatic breast cancer were recruited and scanned for 65 mins after iv injection of 200MBq of GE-226 (mean activity injected for each patient 202MBq (range 164-223MBq, mean radiochemical purity 94%) of radioligand, over one bed position for dynamic imaging, followed by a half-body scan. Blood sampling was used to measure metabolism of the tracer. Safety was assessed. HER2-extracellular domain (ECD) domain was measured in blood. Tumoural uptake was quantified by semi-quantitative and fquantitative parameters in HER2 positive and HER2 negative tumours. Patients had routine baseline FDG imaging. Results: Twenty patients completed the study. GE-226 scans were well tolerated. There were no serious adverse events. GE-226 was slowly metabolised into a single metabolite in the liver; 97% of parent remained at 60 minutes post injection (range 82-100). There was a significant difference in tumoural radioligand uptake between biopsy proven HER2 positive and HER2 negative tumoural patients as measured by SUVmean and SUVmax (p &lt; 0.001). Comparing HER2 positive to HER2 negative cases, there was also a significant difference between tumour to normal tissue uptake ratios SUVmean. Heterogeneous uptake was observed in three patients, two with interlesional uptake variation and one with intralesional heterogeneity. Tumoural uptake increased over time. Normal physiological uptake in salivary glands and the thyroid gland was noted. GE-226 was able to differentiate between lymphadenopathy due to sarcoidosis and cancer in one patient and was superior to FDG which had shown widespread uptake in the benign and malignant nodes. Conclusions: [18F]GE-226 imaging is well tolerated and shows promise for imaging of HER2 positive breast cancer. Further studies with this agent are now planned. Clinical trial information: NCT03827317.