Differentiation of lymphocytes in mouse bone marrow. I. Quantitative radioautographic studies of antiglobulin binding by lymphocytes in bone marrow and lymphoid tissues

The density of surface immunoglobulin on small lymphocytes in the bone marrow and other lymphoid tissues has been compared by radioautographic measurements of antiglobulin binding.Cell suspensions from CBA mice were exposed to ¹²⁵I-labeled rabbit anti-mouse globulin in a wide range of concentrations for 30 min at 0 °C. With increasing concentration of antiglobulin-¹²⁵I the percentage of labeled antiglobulin-binding small lymphocytes in spleen and lymph node suspensions reached well-defined plateau levels. Very few normal or cortisone-resistant thymus cells were labeled under identical conditions. Bone marrow small lymphocytes showed a linear increment in labeled cells throughout the antiglobulin-¹²⁵I dose range, their labeling intensity varied widely, and approximately one half remained unlabeled at high antiglobulin-¹²⁵I concentrations. In 6 wk-old congenitally athymic mice the bone marrow small lymphocyte labeling pattern resembled that in CBA mice, while nearly all (91–97%) small lymphocytes in lymph nodes, thoracic duct lymph and blood, and 75% of those in the spleen, became labeled under plateau conditions. Treatment of cells from 10 wk-old CBA mice with AKR anti-θ C3H serum and complement resulted in almost complete (93%) antiglobulin-labeling of residual small lymphocytes from the spleen but had little effect on bone marrow lymphocyte labeling. Under germfree conditions the proportion of antiglobulin-binding small lymphocytes was slightly elevated in all lymphoid tissues of CBA mice.The results demonstrate that many of the small lymphocytes in mouse bone marrow have readily detectable surface immunoglobulin molecules which vary considerably in density from cell to cell, while others neither have detectable surface immunoglobulin, nor are they θ-bearing, thymus-dependent or recirculating cells. The concept of bone marrow small lymphocytes as a maturing cell population is discussed.     

Comparative radiolabeling and distribution of a tumour-directed monoclonal antibody

The monoclonal antibody (MAb) 155H.7, raised against a synthetic β-anomer of the Thomsen-Friedenreich antigen (S-TAG), was radioiodinated using iodine monochloride, chloramine-T and Iodogen and radiolabeled with ¹¹¹In using the bromoacetamido-derivative of benzyl-EDTA. The in vitro immunoreactivity of the MAb was assessed using an ELISA with the S-TAG and the in vivo distribution of the radioiodinated and radiochelated MAb was determined in the murine mammary carcinoma TA3/Ha tumour model. Both chloramine-T and iodine monochloride radioiodination greatly reduced the immunoreactivity of the MAb compared to radioiodination using lodogen. Bifunctional chelate labeling was comparable to Iodogen in reducing the immunoreactivity of the MAb and subsequent chelation of ¹¹¹In did not further compromise the immunoreactivity of the MAb. The in vivo distribution data showed significantly different distributions of the radiolabels after injection of the radioiodinated and radiochelated MAb. The ¹³¹I-MAb showed some tumour association as compared to the distribution of an ¹²⁵I-non-specific protein and the data also indicates that there is preferential dehalogenation of the radioiodinated MAb. ¹¹¹In from the radiochelated MAb showed significantly higher uptake in the tumour than ¹³¹I from the ¹³¹I-MAb. It is suggested that the differing fates of the two radiolabels within the tumour cell is responsible for the difference in retention observed and not necessarily due to the lack of MAb uptake by the tumour. Overall, the radiochelate label for MAb 155H.7 appears to be superior to radioiodine for in vivo use.     

Multiple occurrence of spontaneous AKR/J lymphomas with T and B cell characteristics.

Three Thy 1.1-positive and surface IgM-positive (Thy 1+, SIg+) AKR/J lymphoma lines are described. These doubly marked tumors arose spontaneously in the peripheral lymphoid organs of 14- to 16-month-old AKR/J mice that either had spontaneous thymus atrophy or had been thymectomized at 1 month of age. All lines bore surface Thy 1.1, Ly,Ig(micron-chain) and Fc receptor (FcR), detectable by immunofluorescence. Immune response region (Iak) antigen was present on the two lines tested. Persistence of Thy 1.1 antigen and SIg after long-term tissue culture provided evidence that these markers were not passively acquired. One of these tumor lines, AkTB-1 always grows in lymph nodes as Thy 1.1-positive,SIg-negative tumors cells, whereas tumor cells growing in the spleen are initially Thy 1.1 positive, SIg negative, but they rapidly acquire SIg, FcR, and 1a between 18 and 21 days of passage.     

N-succinimidyl 5-(trialkylstannyl)-3-pyridinecarboxylates: a new class of reagents for protein radioiodination

N-Succinimidyl 5-(trialkylstannyl)-3-pyridinecarboxylates (alkyl = Me, Bu) have been prepared and used as a precursor to label N-succinimidyl 5-[131I]iodo-3-pyridinecarboxylate (SIPC). SIPC was obtained in greater than 80% yield from either the methyl or butyl precursor with N-chlorosuccinimide and heating at 60-65 degrees C. Significantly lower yields were observed with tert-butyl hydroperoxide. After a 30-min incubation with [131I]SIPC at pH 8.5, goat IgG, an intact monoclonal antibody (MAb), and a MAb F(ab')2 fragment were labeled in 60-65% yield. Specific binding of the MAb and MAb fragment after SIPC labeling was identical with that observed with N-succinimidyl 3-iodobenzoate and higher than that reported previously for these MAbs after labeling by using the Iodogen method. When 5-[131I]iodonicotinic acid was injected into normal mice, thyroid uptake was less than 0.2% of the injected dose, reflecting the inertness of this compound to deiodination. Paired-label biodistribution studies indicate that for both the MAb and the F(ab')2 labeled by using SIPC, accumulation of activity in the thyroid and other tissues is comparable to that observed when these proteins were labeled by using N-succinimidyl 3-iodobenzoate. The results of this study suggest that SIPC may be a reagent for labeling MAbs with halogen nuclides.     

Antigen expression of mouse spleen × thymocyte heterokaryons

The expression of Thy 1.2 and thymic leukemia (TL) antigens by heterokaryons of spleen cells of strain A mice (A-S) and AKR thymocytes (AKR-T) was determined. The A-S parental cells do not express TL antigens, although strain A thymocytes are TL-positive. Approximately 25% of A-S cells express Thy 1.2 antigens; however, AKR-T cells express a different Thy 1 antigen (Thy 1.1) and are phenotypically negative for TL expression. AKR-T × A-S heterokaryons were prepared with the aid of inactivated Sendai virus. Identification of heterokaryons was facilitated by prior isotopic labeling of AKR-T but not A-S cells, and the finding by autoradiography of binucleated cells with one radioactively labeled and one non-labeled nucleus. Antigenic expression of these fused cells was determined by exposure of the cells to specific antiserum and complement prior to autoradiography. 24 hr after fusion, fused cells were resistant to the cytotoxic effects of TL antiserum and fresh complement. However, a large proportion of these cells was lysed by treatment with antiserum directed against the Thy 1.2 antigen.     

SIB-DOTA: A trifunctional prosthetic group potentially amenable for multi-modal labeling that enhances tumor uptake of internalizing monoclonal antibodies

A major drawback of internalizing monoclonal antibodies (mAbs) radioiodinated with direct electrophilic approaches is that tumor retention of radioactivity is compromised by the rapid washout of iodo-tyrosine, the primary labeled catabolite for mAbs labeled via this strategy. In our continuing efforts to develop more versatile residualizing labels that could overcome this problem, we have designed SIB-DOTA, a prosthetic labeling template that combines the features of the prototypical, dehalogenation-resistant N-succinimidyl 3-iodobenzoate (SIB) with DOTA, a useful macrocyclic chelator for labeling with radiometals. Herein we describe the synthesis of the unlabeled standard of this prosthetic moiety, its protected tin precursor, and radioiodinated SIB-DOTA. An anti-EGFRvIII-reactive mAb, L8A4 was radiolabeled with [¹³¹I]SIB-DOTA in 27.1 ± 6.2% (n = 2) conjugation yields and its targeting properties to the same mAb labeled with [¹²⁵I]SGMIB both in vitro and in vivo using U87MG·ΔEGFR cells and xenografts were compared. In vitro paired-label internalization assays showed that the intracellular radioactivity from [¹³¹I]SIB-DOTA-L8A4 was 21.4 ± 0.5% and 26.2 ± 1.1% of initially bound radioactivity at 16 and 24 h, respectively. In comparison, these values for [¹²⁵I]SGMIB-L8A4 were 16.7 ± 0.5% and 14.9 ± 1.1%. Similarly, the SIB-DOTA prosthetic group provided better tumor targeting in vivo than SGMIB over 8 d period. These results suggest that SIB-DOTA warrants further evaluation as a residualizing agent for labeling internalizing mAbs including those targeted to EGFRvIII.     

Radioimmunodetection of human melanoma tumor xenografts with human monoclonal antibodies

We established a human IgM monoclonal antibody that defines a tumor-associated membrane antigen expressed on human melanoma cells. The antigen has been identified as the ganglioside GD2. In this paper, we describe the potential usefulness of the human monoclonal antibody for radioimaging. Nude mice bearing tumors derived from a human melanoma cell line were used as a model. Antibody activity was degradated significantly after labeling with ¹³¹I by the use of a modified chloramine-T method. After testing various concentrations, labeled antibody of a specific activity of 2.8 μCi/ μg produced the best results. Balb/c nude mice bearing a GD2-positive M14 melanoma cell line were injected with 10–30 μg of labeled antibody, and its radiolocalization in different organs and in the whole body were evaluated. The best tumor image was obtained on Day 6. The labeled antibody uptake ratio between tumor and muscle was 9.2:1; the ratio between tumor and liver was 1.4:1. These studies represent the first report of experimental tumor imaging with human monoclonal antibody. Human monoclonals will probably prove to be superior reagents for tumor imaging in melanoma patients if the problem of antibody radiolysis is resolved.     

The mitogenic properties of rabbit anti-mouse brain serum

The mitogenic activity of heterologous rabbit anti-mouse brain sera (RAMB) was investigated. By complement-dependent cytotoxicity and indirect immunofluorescence, RAMB was T-cell specific. Mitogenic activity was assessed by determination of [³H]thymidine incorporation into DNA. RAMB was mitogenic for spleen cells for Thy 1.1- and Thy 1.2-positive mouse strains. Maximal mitogenic responsiveness to RAMB occurred on Day 3 of culture. The incorporation of [³H]uridine into RNA and [³H]leucine into protein and percentage of blast cells in culture were also significantly increased following RAMB stimulation. The mitogenic activity of RAMB was abrogated by adsorption of the sera with BALB/c or AKR thymocytes or brains or with RL♂ 1.3+, a Thy 1.2-bearing T-cell lymphoma of BALB/c origin. In contrast, the mitogenic activity was not removed when RAMB sera were absorbed with RL♂ 1.4?, a variant of RL♂ 1 which appears to specifically lack cell surface Thy 1 determinants. These data suggest that the mitogenic activity of RAMB is Thy 1 directed. RAMB mitogenicity is T-cell dependent. Spleen cells from normal and heterologous nu/+ mice respond to RAMB, while spleen cells from nu/nu mice do not respond. Normal thymocytes and cortisone-resistant thymocytes do not respond mitogenically to RAMB. The response of unseparated spleen cells to RAMB is also macrophage dependent. Nylon-wool column-purified splenic T cells respond to high concentrations of RAMB in the absence of exogenous macrophages but do not respond to lower concentrations of RAMB unless exogenous macrophages are added to the cultures. Nylon-wool-adherent cells, which are B-cell enriched and relatively T-cell depleted, also respond to RAMB, suggesting that in the presence of even small numbers of T cells, B cells can be recruited into the response.     

Identification of subsets of proliferating low Thy 1 cells in thymus cortex and medulla

Subsets of proliferating thymocytes were identified in the normal mouse thymus by in vivo labeling with [³H]TdR and by cell separation according to relative amounts of Thy 1 antigen. In order to resolve apparent discrepancies in the literature, parenteral and topical application of [³H]TdR were compared as labeling methods for dividing thymocytes, and limited complement lysis and fluorescence-activated cell sorting were compared as separation principles for high Thy 1 and low Thy 1 thymocyte subsets. The separated cells were further characterized by immunofluorescence for terminal deoxynucleotidyltransferase (TdT), which normally is restricted to cortical thymocytes, and for H2 alloantigens, which are preponderant on medullary thymocytes. Four subsets of proliferating cortical thymocytes were identified after application of [³H]TdR to the thymus capsule. The major subset, which comprised about 92% of dividing cortical thymocytes, had a high Thy 1, low H2 phenotype. Most were also TdT + ve. The three minor subsets of proliferating cortical thymocytes each had a low Thy 1 phenotype, but differed according to H2 and TdT markers. Systemic injection of [³H]TdR also labeled the above subsets of dividing cortical thymocytes, but in addition it detected a subset of proliferating low Thy 1, low H2, TdT — ve cells in the thymus medulla. The latter subset comprised about one-third of the pool of proliferating low Thy 1 cells. In their aggregate the four subsets of low Thy 1 cells constituted approximately 13% of total proliferating thymocytes and 1.1% of total thymocytes. The identification of discrete subsets of proliferating low Thy 1 cells in the thymus cortex as well as in the thymus medulla is compatible with the hypotheses that all thymocytes are descended from low Thy 1 precursors and that separate precursor cell subsets exist for cortical and medullary thymocytes.     

An improved method of direct labeling monoclonal antibodies with 99mTc

An improved method of direct labeling MAbs with ^99mTc is described. Two murine monoclonal antibodies, designated Lym-1 and B72.3, have been successfully labeled with ^99mTc in 0.1 M borate buffer at pH 9.3. The choice of buffer and pH was essential for obtaining a radiolabeling yield ⩾98%. In vitro studies demonstrated that the radiolabeled antibodies were stable and retained their immunoreactivity. Imaging and biodistribution studies using Raji and LS174T human tumor-bearing nude mice demonstrated a significant tumor uptake at 24-h post-injection of ^99mTc-labeled MAbs. This improved labeling method showed better stability than those of previously published methods and resulted in significant improvement in the uptake of antibody in tumor. External images at 24 h post-injection revealed clearly visible tumors demonstrating the benefit of this method for tumor immunoscintigraphy.     

Targeting of anti-Thy 1.1 monoclonal antibody conjugated liposomes in Thy 1.1 mice after intraveneous administration

¹²⁵I-labeled liposomes, conjugated to an anti-Thy 1.1 monoclonal antibody (MRCOX7), demonstrated up to 7.4-fold greater lymph node uptake than liposomes conjugated to non-specific monoclonal antibody (R-10) after intravenous injection into Thy 1.1 (AKR-J) mice. Uptake of anti-Thy 1.1-conjugated liposomes by the lymph nodes of AKR-J mice was 3-times greater than their uptake by lymph nodes of Thy 1.2 (AKR-Cu) mice. Lymph node localization of anti-Thy 1.1-liposomes was equal to that of control monoclonal antibody-liposomes in Thy 1.2 mice. Conjugation to either monoclonal antibody substantially increased liposome clearance by the liver, while decreasing liposome uptake in a number of organs outside the reticuloendothelial system. Changes in liposome size and phospholipid composition did not significantly alter these results. Administration of a large predose of unconjugated liposomes prior to injection of MRCOX7-conjugated liposomes increased blood levels and reduced liver uptake of the monoclonal antibody-liposome conjugates, but did not further enhance lymph node uptake. This study demonstrates that targeting of liposomes by conjugation to the appropriate monoclonal antibody, can significantly increase their uptake in lymph nodes which contain high levels of cells expressing the target antigen. However, conjugation to monoclonal antibody also increases clearance of liposomes by the liver. To increase the uptake of monoclonal antibody-conjugated liposomes in target tissue, substantial reduction of their clearance by the reticuloendothelial system will be required.     

Immunoscintigraphy with biosynthetically-labeled 75Se-antibodies—II. Comparison with antibodies labeled by radioiodination

The biodistributions of ⁷⁵Se and ¹²⁵I were compared in nude mice bearing melanomas and injected simultaneously with biosynthetically-labeled ⁷⁵Se-MoAbs and radioiodinated ¹²⁵I-MoAbs from the same anti-melanoma hybridoma. The ⁷⁵Se dose recovery in the tumor (day 10) was twice that for ¹²⁵I, but the tumor to normal tissue ratios were identical. It is concluded that biosynthetically-labeled MoAbs survive better in vivo, while the tumor to normal tissue ratios are governed by the excretion rate of labeled MoAb catabolytic products.     

Protein radiohalogenation: observations on the design of N-succinimidyl ester acylation agents

In previous studies we have demonstrated that antibodies radioiodinated with N-succinimidyl 3-iodobenzoate (SIB) are less susceptible to loss of radioiodine in vivo than antibodies iodinated directly by electrophilic substitution on their tyrosine residues with Iodogen. Since the Bolton-Hunter reagent, N-succinimidyl 3-(4-hydroxy-3-iodophenyl)propionate, is identical with SIB except that it contains a hydroxyl group on the aromatic ring and a two-methylene spacer, a comparison of their coupling chemistry and in vivo behavior was performed to better understand the structural requirements for a useful iodinated acylation agent. Protein concentration and pH had a significant effect on the coupling efficiency of both SIB and the Bolton-Hunter reagent; however, protein-labeling yields with SIB were generally higher by a factor of 2. Paired-label biodistribution studies in mice demonstrated that thyroid uptake (a monitor of dehalogenation) of antibody labeled by the Bolton-Hunter method was twice that of antibody labeled with SIB but only 7% of that observed for antibody labeled with Iodogen. These results suggest that even minor differences in iodination site can profoundly alter the retention of label on a protein in vivo.     

Non-dehalogenation mechanisms for excretion of radioiodine after administration of labeled antibodies

In patients or mice with cancer the pharmacokinetic behavior of radioiodinated and radiometal chelated antibodies has been observed to be different. Rapid clearance from the tissues and excretion into the urine can occur after injection of radioiodinated antibodies. These observations have been interpreted to reflect in vivo dehalogenation of the antibody. This publication describes a variety of other mechanisms that can underlie these phenomena. These mechanisms include receptor uptake and catabolism of antibody and instability of the labeled antibody due to the labeling conditions. Specifically, the relative masses of chloramine-T and antibody in the iodination reaction mixture, the level of iodination of the antibody, and the amount of antibody administered to the recipient are all factors which can influence the clearance of radioiodinated antibody from the recipient. The final determinant for the different behavior of radioiodinated and In-111 metal chelated antibody relate to the different biologic pathways of indium when compared to iodine.     

Radioiodinated iodobenzoyl conjugates of a monoclonal antibody Fab fragment. In vivo comparisons with chloramine-T-labeled Fab

A comparative investigation of the biodistributions of radioiodinated p- and m-iodobenzoyl conjugates of a monoclonal antibody Fab fragment, NR-LU-10 Fab, and the same antibody Fab fragment radioiodinated by the chloramine-T (ChT) method has been carried out in mice. Coinjected, dual-isotope studies in athymic mice with tumor xenografts have demonstrated that there are only minor differences in the in vivo distributions of the iodobenzoyl-labeled Fabs, except in the excretory organs, kidneys, and intestines, where major differences were observed. Similarly, coinjection of either the p-iodobenzoyl or m-iodobenzoyl conjugate of NR-LU-10 Fab with the Fab radioiodinated with ChT/radioiodide into BALB/c mice provided additional data that indicated that the two iodobenzoyl conjugates distributed similar in a number of selected tissues. The tissue-distribution differences of the regioisomeric iodobenzoyl conjugates in relation to the ChT-radioiodinated Fab were large for the stomach and neck, consistent with previous studies. The most notable difference between the two iodobenzoyl conjugates was the kidney activity, where the m-iodobenzoyl conjugate was similar to the directly labeled Fab, but the p-iodobenzoyl-conjugated Fab was higher by nearly a factor of 2.     

Resistance to transplantation of spontaneous AKR lymphoma cells by substrains of AKR mice. In vivo resistance and generation of an H-2 restricted CML response.

High leukemia incidence AKR/J (H-2k, Thy 1.1) and AKR/Cu (H-2k, Thy 1.2) substrains of AKR mice reject the reciprocal strains spontaneously arising lymphoma cells. In the course of rejection, splenic precursor cells are generated which, upon secondary stimulation in vitro, result in specific cytotoxic T-cells. The antigenic component of AKR cells resulting in this secondary CML response resides on both lymphoma and normal T- and B-cells, and is distinct from the Thy antigen. Primed AKR/J lymphocytes will respond to and only lyse immunogens and targets homologous at the K- and-or D-end of the MHC, while primed AKR/Cu cells require homology at the D-end of the MHC.     

Hymenolepis microstoma: Mouse strain differences in resistance to a challenge infection

Antibody responses and host resistance to the tapeworm, Hymenolepis microstoma, were investigated using $\text{AKR}\text{J}$ and $\text{C}\text{3}\text{HeB}\text{FeJ}$ strains of mice. AKR mice were significantly more resistant than controls to a secondary infection following exposure to a 3-, 21-, or 40-day primary infection. During a primary infection, intestinal anti-worm antibody responses measured by an enzyme-linked immunosorbent assay were elevated in the more resistant AKR strain, whereas serum antibody titers did not differ between the two strains. However, during a secondary infection, serum IgA titers were higher in AKR mice than C3H mice. Suppression of the serum IgA anti-worm response by oral administration of lipopolysaccharide also suppressed resistance to a secondary infection. Intraperitoneal immunization with worm antigen resulted in a minor degree of protection in AKR mice. This protection was associated with increased intestinal antibody titers compared to mice not demonstrating protection. These results suggest that the protective responses observed in AKR mice relative to C3H mice reflect differences in mucosal antibody responses to H. microstoma.     

Labeling and stability of radiolabeled antibody fragments by a direct 99mTc-labeling method

The in vitro labeling and stability of ^99mTc-labeled antibody Fab′ fragments prepared by a direct labeling technique were evaluated. Eight antibody fragments derived from murine IgG1 (N = 5), IgG2a (N = 2) and IgG3 (N = 1) isotypes were labeled with a preformed ^99mTc-d-glucarate complex. No loss of radioactivity incorporation was observed for all the ^99mTc-labeled antibody fragments after 24 h incubation at 37 °C. The ^99mTc-labeled antibody fragments (IgG1, N = 2; IgG2a, N = 2; IgG3, N = 1) were stable upon challenge with DTPA, EDTA or acidic pH. Furthermore, using the affinity chromatography technique, two of the ^99mTc-labeled antibody fragments displayed no loss of immunoreactivity after prolonged incubation in phosphate buffer up to 24 h at 37 °C. The bonding between ^99mTc and antibody fragments was elucidated by challenging with a diamide ditholate (N₂S₂) compound. The Fab′ with IgG2a isotype displayed tighter binding to ^99mTc in comparison to the Fab′ from IgG1 and IgG3 isotype in N₂S₂ challenge and incubation with human plasma. The in vivo biodistribution of five ^99mTc-labeled fragments were evaluated in normal mice. In conclusion, the direct labeling method allows stable ^99mTc labeling of antibody fragments from three of the major murine isotypes.     

Strategies for enhancement of radioimmunotherapy

Two new strategies for increasing tumor uptake have been investigated. First the effect of interleukin-2 (IL-2) on tumor uptake of ¹²⁵I-Lym-l antibody in nude mice was investigated. Secondly, the use of ⁶⁷Cu-labeled Lym-1 was evaluated in patients. In nude mice implanted with Raji human lymphoma, a greater than 2-fold enhancement of tumor uptake of ¹²⁵I-Lym-1 was observed after administration of PEG-interleukin-2 (PEG-IL-2). The macrocycle 1,4,8,11-tetraazacylcotetradecane-N,N′,N′,N'''-tetraacetic acid (TETA), synthesized specifically for copper chelation, has been conjugated to Lym-1 for ⁶⁷Cu labeling of the monoclonal antibody (MoAb). There was no evidence for bone or normal marrow uptake and the residence time in the tumor was prolonged. Surprisingly, a dose of 4.4 mCi that was intended for imaging induced substantial tumor regression in a patient.     

Preparation and Identification of HER2 Radioactive Ligands and Imaging Study of Breast Cancer-Bearing Nude Mice

OBJECTIVE: A micro-molecule peptide TP1623 of ^99mTc-human epithelial growth factor receptor 2 (HER2) was prepared and the feasibility of using it as a HER2-positive molecular imaging agent for breast cancer was evaluated. METHODS: TP1623 was chemically synthesized and labeled with ^99mTc. The labeling ratio and stability were detected. HER2 expression levels of breast cancer cells (SKBR3 and MDA-MB-231) and cell binding activity were measured. Biodistribution of ^99mTC-TP1623 in normal mice was detected. SKBR3/MDA-MB-231-bearing nude mice models with high/low expressions of HER2 were established. Tumor tissues were stained with hematoxylin–eosin (HE) and measured by immunohistochemistry to confirm the formation of tumors and HER2 expression. SPECT imaging was conducted for HER2-overexpressing SKBR3-bearing nude mice. The T/NT ratio was calculated and compared with that of MDA-MB-231-bearing nude mice with low HER2 expression. The competitive inhibition image was used to discuss the specific binding of ^99mTc- TP1623 and the tumor. RESULTS: The labeling ratio of ^99mTc-TP1623, specific activity, and radiochemical purity (RCP) after 6 h at room temperature were (97.39 ± 0.23)%, (24.61 ± 0.06) TBq/mmol, and (93.25 ± 0.06)%, respectively. HER2 of SKBR3 and MDA-MB-231 cells showed high and low expression levels by immunohistochemistry, respectively. The in vitro receptor assays indicated that specific binding of TP1623 and HER2 was retained. Radioactivity in the brain was always at the lowest level, while the clearance rate of blood and the excretion rate of the kidneys were fast. HE staining showed that tumor cells were observed in SKBR3- and MDA-MB-231-bearing nude mice, with significant heteromorphism and increased mitotic count. The imaging of mice showed that targeted images could be made of ^99mTc-TP1623 in high HER2-expressing tumors, while no obvious development was shown in tumors in low HER2-expressing nude mice. No development was visible in tumors in competitive inhibition of imaging, which indicates the combination of ^99mTc-TP1623 and tumor was mediated by HER2. CONCLUSION: High labeling ratio and specific activity of ^99mTc-TP1623 is successfully prepared; it is a molecular imaging agent for HER2-positive tumors that has potential applicative value.