CD38‐Specific Gallium‐68 Labeled Peptide Radiotracer Enables Pharmacodynamic Monitoring in Multiple Myeloma with PET

Abstract

The limited availability of molecularly targeted low‐molecular‐weight imaging agents for monitoring multiple myeloma (MM)‐targeted therapies has been a significant challenge in the field. In response, a first‐in‐class peptide‐based radiotracer, [⁶⁸Ga]Ga‐AJ206, is developed that can be seamlessly integrated into the standard clinical workflow and is specifically designed to noninvasively quantify CD38 levels and pharmacodynamics by positron emission tomography (PET). A bicyclic peptide, AJ206, is synthesized and exhibits high affinity to CD38 (K _D: 19.1 ± 0.99 × 10⁻⁹ m) by surface plasmon resonance. Further, [⁶⁸Ga]Ga‐AJ206‐PET shows high contrast within 60 min and suitable absorbed dose estimates for clinical use. Additionally, [⁶⁸Ga]Ga‐AJ206 detects CD38 expression in cell line‐derived xenografts, patient‐derived xenografts (PDXs), and disseminated disease models in a manner consistent with flow cytometry and immunohistochemistry findings. Moreover, [⁶⁸Ga]Ga‐AJ206‐PET successfully quantifies CD38 pharmacodynamics in PDXs, revealing increased CD38 expression in the tumor following all‐trans retinoic acid (ATRA) therapy. In conclusion, [⁶⁸Ga]Ga‐AJ206 exhibits the salient features required for clinical translation, providing CD38‐specific high‐contrast images in multiple models of MM. [⁶⁸Ga]Ga‐AJ206‐PET could be useful for quantifying total CD38 levels and pharmacodynamics during therapy to evaluate approved and new therapies in MM and other diseases with CD38 involvement.

A novel peptide‐based gallium‐68 labeled radiotracer, [⁶⁸Ga]Ga‐AJ206, is developed for imaging multiple myeloma using positron emission tomography (PET). [⁶⁸Ga]Ga‐AJ206 delivers PET images with high contrast within 60 min and detected varying levels of CD38 expression with high sensitivity (left panel). Furthermore, [⁶⁸Ga]Ga‐AJ206‐PET enables noninvasive quantification of CD38 pharmacodynamics (right panel).

1. Introduction

Multiple myeloma (MM) is a plasma cell neoplasm that primarily arises in the bone marrow.^{[ 1 ]} Clinically, MM is characterized by wide variability in response to therapy and survival thought to be due to biologic heterogeneity in both cell‐intrinsic and ‐extrinsic factors.^{[ 1 ]} Therapeutic options for MM include combinations of drugs with different mechanisms of action, such as proteasome inhibitors (PIs), immunomodulatory drugs (IMIDs), B cell maturation antigen (BCMA) targeting agents, monoclonal antibodies targeting MM antigens (mAbs, including bispecific monoclonal antibodies),^{[ 2 ]} chimeric antigen receptor T cells (CAR‐T), and high‐dose chemotherapy rescued by autologous stem cell transplantation (ASCT). Current methods to quantify the burden of MM in patients include bone marrow biopsies, serum testing of monoclonal paraprotein, and cross‐sectional imaging.^{[ 3} , ^{4 ]} Among the latter, positron emission tomography combined with computed tomography (PET‐CT) with the metabolic radiotracer 2‐deoxy‐2‐[¹⁸F]fluoro‐D‐glucose ([¹⁸F]FDG) is the best equipped to determine response to treatment and provide prognostic information.^{[ 5 ]} The significant limitations of [¹⁸F]FDG include a lack of specificity due to uptake in inflammatory cells.^{[ 6 ]} Also, some cases of myeloma are not FDG‐avid.^{[ 7 ]} Thus, the development of molecularly targeted PET imaging agents that enhance MM detection specificity, sensitivity, and early therapeutic response assessment remains an unmet need.

Cluster of differentiation protein 38 (CD38) is expressed uniformly and with high density in MM. Taking advantage of high CD38 expression, several therapies including antibodies, antibody‐drug conjugates, and CAR‐T cells have been developed or are in different phases of clinical development.^{[ 8 ]} Continuous therapy with a combination of the aforementioned agents has been associated with the best outcomes in MM.^{[ 9 ]} However, there are efforts underway to reduce toxicity by tailoring or tapering treatment in patients who have little or no detectable measurable residual disease (MRD).^{[ 10 ]} As a result, accurate detection of small amounts of disease will become increasingly important for this growing patient population. Also, efforts are underway to increase CD38 expression on MM cells by treatment with agents such as all‐trans retinoic acid (ATRA) to take advantage of the excellent anti‐CD38 therapeutic options available.^{[ 11} , ^{12 ]} However, options to monitor those therapies in real‐time have been few. Realizing the need to improve specificity and sensitivity to detect MM, several groups have leveraged the high expression of CD38 on MM cells^{[ 13} , ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸ , ¹⁹ , ²⁰ , ²¹ , ^{22 ]} to develop molecularly targeted imaging agents. Radiolabeled conjugates of anti‐CD38 antibodies have been shown to detect MM in preclinical models and patients, with superior specificity and sensitivity compared to the routinely used [¹⁸F]FDG‐PET.^{[ 13} , ^{19 ]} However, the long biological half‐life of those antibody‐based imaging agents limits their applicability to patients who have received anti‐CD38 therapy. Additionally, although the diagnostic and therapeutic implications are less‐well understood, CD38 is expressed in other cancer types including prostate cancer, and molecularly targeted agents may have a more pan‐cancer role in the long term.

Peptides are gaining traction as effective tools for targeting cancer due to their high specificity, rapid clearance, and ease of synthesis.^{[ 23 ]} In particular, peptide‐radiotracers are utilized to improve contrast and enhance the detection of cancerous lesions.^{[ 24 ]} Compared to antibodies, peptides have shorter clearance times (hours instead of days), making them a favorable option for repeated imaging for assessing response to therapies. The tractable pharmacokinetic properties of peptide‐based imaging agents also enable the quantification of target pharmacodynamics as we have shown for imaging programmed death‐ligand 1 (PD‐L1) expression in cancers.^{[ 25} , ^{26 ]} Notably, the application of low molecular weight agents for targeting CD38 in MM has not been previously reported. In this study, we capitalized on these characteristics to develop a first‐in‐class peptide‐based low molecular weight agent that allows for the quantification of CD38 levels and pharmacodynamics throughout the body within a few hours.

Here, we report the development and evaluation of a new gallium‐68 labeled peptide‐based radiotracer, [⁶⁸Ga]Ga‐AJ206, for CD38 targeting in MM. We assessed the pharmacokinetics, biodistribution, and CD38 specificity of [⁶⁸Ga]Ga‐AJ206 in vitro and in vivo using multiple MM cell lines with variable CD38 levels, primary MM cells and their xenografts, and in disseminated tumor models. Additionally, we investigated the potential of [⁶⁸Ga]Ga‐AJ206‐PET to detect the pharmacodynamics of CD38 during all‐trans retinoic acid (ATRA) therapy.

2. Results

2.1. Synthesis and Characterization of AJ206

CD38‐binding low‐molecular‐weight imaging agents have not been reported and high‐affinity peptide therapeutics (IC₅₀ < 100 × 10⁻⁹ m) were reported only recently, which form the basis for our imaging agents (Figure  1 ).^{[ 27 ]} To test the applicability of those peptides for in vivo imaging, we selected a peptide with high hydrophilicity and a free amine for further functionalization. We synthesized a 2,2′,2″‐(1,4,7‐triazacyclononane‐1,4,7‐triyl)triacetic acid (NOTA)‐conjugated peptide, AJ206, with a poly ethylene glycol (PEG) linker to improve solubility (Figure 1A; Schemes S1 and S2, Supporting Information). The agent was characterized by mass spectrometry (Figure S1, Supporting Information). We then tested its potential to bind human CD38 (hCD38) and mouse CD38 (mCD38) recombinant proteins and MM cells with variable CD38 expression. AJ206 exhibited high affinity for hCD38 (will be referred as CD38) with a dissociation constant (K _D) of 19.1 × 10⁻⁹ m by surface plasmon resonance (SPR) but did not bind mCD38 (Figure 1B and Figure S2, Supporting Information).

Figure 1.

Structure and in vitro characterization of AJ206. A) Structure of bicyclic peptide AJ206 having NOTA as bifunctional chelator for ⁶⁸Ga‐labeling B) Surface plasmon resonance (SPR) analysis showing affinity of AJ206 for CD38 using recombinant human CD38 protein; data is represented as mean ± SEM (n = 2).

2.2. Pharmacokinetics of [⁶⁸Ga]Ga‐AJ206

We synthesized the radioactive analog of AJ206, [⁶⁸Ga]Ga‐AJ206, by standard ⁶⁸Ga‐radiolabeling chemistry (Scheme S3, Supporting Information) to evaluate the pharmacokinetics, biodistribution, and in vivo specificity.^{[ 28 ]} [⁶⁸Ga]Ga‐AJ206 was obtained with decay corrected radiochemical yields of 92 ±10.5% (n = 35) with >95% radiochemical purity and a specific activity (molar activity) of 10–15 GBq µmol⁻¹ (270–400 mCi µmol⁻¹) (Figure S3, Supporting Information). The formulated [⁶⁸Ga]Ga‐AJ206 dose remained stable for 2 h (Figure S4, Supporting Information) and exhibited an octanol/PBS partition coefficient (LogD, pH‐7.4) of −1.32 ± 0.08. Using immunodeficient NSG mice with MOLP8 human MM xenografts that are known to express CD38,^{[ 29 ]} we evaluated [⁶⁸Ga]Ga‐AJ206 in vivo. Whole body dynamic PET/MR images of those mice acquired over 90 min showed clear and high radiotracer accumulation in tumors as early as few minutes after injection, with a higher tumor contrast observed at 60 min due to radiotracer clearance (Figure  2A). Kidney and bladder tissues had the highest radiotracer accumulation among normal tissues, consistent with the renal clearance mechanism for low‐molecular‐weight peptides, while radioactivity from other tissues, including liver, declined over 60 min (Figure 2B). These imaging data showed that [⁶⁸Ga]Ga‐AJ206 exhibits in vivo kinetics and contrast desirable for an imaging agent. To verify the PET results, we conducted ex vivo biodistribution studies (Table  1 and Table S1, Supporting Information). At 60 min, [⁶⁸Ga]Ga‐AJ206 uptake in MOLP8 tumors peaked at 3.76 ± 0.25% %ID g⁻¹ (Figure 2C). Blood pool activity exhibited consistent washout, with almost 95% of activity cleared by 60 min, and comparable radioactivity washouts were observed in all other non‐specific tissues. As a result, the tumor‐to‐blood and tumor‐to‐muscle ratios were highest between 60 and 120 min (Figure 2D and Table 1). Thus, we chose the 60 min time point for all further experiments because it provided good contrast and aligned with the standard PET imaging time frame.

Figure 2.

Pharmacokinetics of [⁶⁸Ga]Ga‐AJ206 in NSG mice bearing MOLP8 tumor xenografts. A) Coronal sections of the fused dynamic PET/MR images showing [⁶⁸Ga]Ga‐AJ206 distribution. Primary tumor is indicated by the dashed white circle. Mice were intravenously injected with ≈ 9.25 MBq (≈250 µCi) [⁶⁸Ga]Ga‐AJ206; L, Liver; B, Bladder. B) Time–activity curves of [⁶⁸Ga]Ga‐AJ206 in the kidney, heart, liver, tumor and muscle derived from images A. Data in panel B is represented as mean ± SEM (n = 3). C) Uptake of [⁶⁸Ga]Ga‐AJ206 in tumor, blood, liver and muscle derived from ex vivo biodistribution study. D) Tumor‐to‐muscle and tumor‐to‐blood ratios derived from biodistribution data. Data in panels C and D are from mice intravenously injected with ≈ 2.96 MBq (≈80 µCi) [⁶⁸Ga]Ga‐AJ206 and sacrificed at different time‐points after injection, represented as mean ± SEM (n = 4 or 5).

Table 1.

Kinetics and distribution of [⁶⁸Ga]Ga‐AJ206 in mice with MOLP8 tumor xenografts; data is presented as mean ± SEM (n = 4 or 5) of %ID g⁻¹.

Tissues	5 min	30 min	60 min	120 min
Blood	8.25 ± 0.50	2.07 ± 0.30	0.56 ± 0.09	0.05 ± 0.01
Muscle	0.92 ± 0.11	0.37 ± 0.05	0.14 ± 0.02	0.07 ± 0.04
Tumor	2.58 ± 0.18	2.91 ± 0.84	3.76 ± 0.25	1.46 ± 0.02
Heart	3.13 ± 0.27	0.83 ± 0.18	0.29 ± 0.03	0.05 ± 0.01
Lungs	6.16 ± 0.16	1.86 ± 0.29	0.71 ± 0.10	0.16 ± 0.03
Liver	21.24 ± 1.31	6.39 ± 3.71	1.22 ± 0.09	0.42 ± 0.01
Spleen	2.28 ± 0.50	0.75 ± 0.14	0.39 ± 0.03	0.17 ± 0.02
Kidney	11.17 ± 0.65	17.09 ± 1.77	16.65 ± 2.52	13.75 ± 0.23
Stomach	1.08 ± 0.07	0.51 ± 0.06	0.25 ± 0.06	0.03 ± 0.01
Small intestine	1.95 ± 0.08	0.59 ± 0.14	0.65 ± 0.09	0.18 ± 0.04
Femur	1.42 ± 0.09	0.65 ± 0.08	0.28 ± 0.07	0.07 ± 0.01
Tumor/muscle	2.94 ± 0.47	7.28 ± 1.89	27.12 ± 2.73	27.37 ± 18.55
Tumor/blood	0.32 ± 0.04	1.43 ± 0.46	7.20 ± 0.89	19.72 ± 6.57

2.3. Human Radiation Dosimetry Estimates

Pharmacokinetic data obtained from biodistribution studies of MM1S tumor‐bearing mice were used to predict the time‐integrated activity coefficients (TIACs, previously known as residence times) of [⁶⁸Ga]Ga‐AJ206 in humans as described previously^{[ 30} , ^{31 ]} (Tables S1 and S2, Supporting Information). TIAC estimates were then employed as input to MIRDCalc to calculate the absorbed dose coefficients to organs from ⁶⁸Ga using the ICRP adult female reference phantom (Table S2, Supporting Information). The kidneys received the highest absorbed dose (0.13 rem per mCi), followed by the liver (0.06 rem per mCi), heart wall (0.05 rem per mCi), and lung (0.05 rem per mCi). Based on these results, a 30 mCi dosage can be safely administered, with an estimated effective dose equivalent of less than 5 rem, to obtain PET images.

2.4. Confirmation of [⁶⁸Ga]Ga‐AJ206 Specificity in MM Cell Lines and Xenografts with Varying CD38 Levels

Next, we conducted an evaluation of the specificity of [⁶⁸Ga]Ga‐AJ206 to accurately detect varying levels of CD38 expression both in vitro and in vivo. Four MM cell lines (U266, RPMI, MM1S, and MOLP8) were incubated with [⁶⁸Ga]Ga‐AJ206, and the amount of cell‐bound radioactivity was measured. We observed that MOLP8 and U266 cells showed the highest and the least amount of radioactivity binding, respectively, while RPMI and MM1S cells demonstrated intermediate uptake (Figure  3A). We confirmed the specificity of [⁶⁸Ga]Ga‐AJ206 binding by observing a significant reduction in radiotracer uptake (P < 0.0001) when cells were incubated with a saturating concentration (2 × 10⁻⁶ m) of nonradioactive AJ206. To validate the in vitro uptake, we performed flow cytometry analysis to measure cell surface expression and density, and western blot analysis to assess the total CD38 protein expression. Our results showed that the highest cell surface receptor expression and density were observed in MOLP8 cells followed by RPMI and MM1S cells, with the lowest expression in U266 cells (Figure 3B,C). Similarly, MOLP8 cells exhibited the highest total CD38 expression, followed by RPMI and MM1S cells, while U266 cells had the lowest (Figure 3D). We also performed a correlation analysis to investigate the relationship between [⁶⁸Ga]Ga‐AJ206 uptake and CD38 receptor density, and found a strong correlation (R² = 0.9511), indicating that [⁶⁸Ga]Ga‐AJ206 uptake in cells is CD38 specific (Figure 3E). Furthermore, to evaluate internalization potential of the peptide, RPMI cells were incubated with [⁶⁸Ga]Ga‐AJ206 and internalized activity was measured by treating the cells with acidic buffer (pH 3.2) and measuring the internalized activity. The resulting data showed a continuous increase in intracellular accumulation of [⁶⁸Ga]Ga‐AJ206, indicating internalization of radiotracer bound receptor (Figure S5, Supporting Information).

Figure 3.

In vitro specificity of [⁶⁸Ga]Ga‐AJ206 for CD38. A) [⁶⁸Ga]Ga‐AJ206 binding (percent incubated activity, %IA) to different MM cells. Cells were incubated with 1 µCi [⁶⁸Ga]Ga‐AJ206 at 4 °C for 1 h. [⁶⁸Ga]Ga‐AJ206 uptake is CD38 expression dependent, and co‐incubation with 2 × 10⁻⁶ m of nonradioactive AJ206 (blocking dose) significantly reduced radiotracer uptake confirming CD38 specificity. B) Flow cytometry analysis of CD38 surface expression in MM cells. C) CD38 receptor density in MM cells measured by quantibrite assay. D) Representative western blot of total CD38 protein expression (bottom panel). Densiometric analysis of western blot preformed using ImageJ software and band intensities represented as a ratio of CD38 protein to GAPDH control (top panel). E) Correlation of [⁶⁸Ga]Ga‐AJ206 uptake with surface CD38 receptor density; data in panels A, C, and E are represented as mean ± SD (n = 3‐4). ns, P ≥ 0.05; ****, P ≤ 0.0001 is by unpaired Student's t test. Simple linear regression and Pearson coefficient were used in E.

To validate the in vitro results, we performed PET imaging studies of MM xenografts derived from the cell lines mentioned above, using [⁶⁸Ga]Ga‐AJ206. PET images were acquired at 60 min and showed highest radioactivity accumulation in MOLP8 tumors, followed by MM1S and RPMI tumors, and least accumulation in U266 tumors (Figure  4A). To corroborate the PET data, we performed IHC staining of the same xenografts for CD38 and found intense staining in MOLP8 xenografts and least in U266 tumors (Figure 4B and Figure S7 Supporting Information). Additionally, we conducted ex vivo biodistribution studies to further validate the imaging findings. We found that the tumor uptake was consistent with in vitro and PET results, with the highest uptake observed in MOLP8 tumors (3.76 ± 0.25%ID g⁻¹), followed by MM1S (2.48 ± 0.17%ID g⁻¹) and RPMI (1.7 ± 0.36). On the other hand, U266 tumors showed 0.25 ± 0.03%ID g⁻¹ of [⁶⁸Ga]Ga‐AJ206, which is in line with their low CD38 expression. The tumor‐to‐muscle and tumor‐to‐blood ratios showed similar trends with respect to CD38 status (Figure 4C), and nonspecific tissues showed no significant differences between tumor models (Table S3, Supporting Information). Furthermore, we demonstrated the specificity of [⁶⁸Ga]Ga‐AJ206 for CD38 by co‐administering 2 mg kg⁻¹ nonradioactive AJ206, which significantly reduced the radioactivity uptake in MM1S tumor xenografts (P < 0.01) (Figure 4D,E), however no significant difference was observed in nonspecific tissues (Figure S6, Supporting Information). These in vitro and in vivo data together demonstrate the potential of [⁶⁸Ga]Ga‐AJ206 for noninvasive detection of varying levels of CD38 expression.

Figure 4.

In vivo specificity of [⁶⁸Ga]Ga‐AJ206 for CD38 in NSG mice with MM tumor xenografts. A) Whole‐body PET/CT images of different human MM xenografts at 60 min after the injection of radiotracer. Mice were injected with ≈ 7.4 MBq (≈200 µCi) [⁶⁸Ga]Ga‐AJ206. B) IHC staining for CD38 expression in MM xenografts. C) [⁶⁸Ga]Ga‐AJ206 uptake quantification (%ID g⁻¹) in different MM tumors by ex vivo biodistribution at 60 min after injection. D) PET/CT images of MM1S tumor xenograft‐bearing mice with [⁶⁸Ga]Ga‐AJ206, with and without pre‐administration of a blocking dose (2 mg kg⁻¹ of AJ206) (tumor denoted with dashed red line). E) [⁶⁸Ga]Ga‐AJ206 quantification in tumors by ex vivo biodistribution in mice treated with and without a blocking dose; data in figure C and E are shown as box and whisker plots showing all data points (n = 4–5). Ordinary one‐way ANOVA using multiple comparison test in C and multiple unpaired t test in E. ns, P ≥ 0.05; *, P ≤ 0.05; ** P ≤ 0.01; ***, P ≤ 0.001.

2.5. Validation of [⁶⁸Ga]Ga‐AJ206 Specificity for CD38 in Disseminated MM Disease Models and Primary Plasma Cell Leukemia Xenografts

We next conducted a study to evaluate the potential of [⁶⁸Ga]Ga‐AJ206 to visualize disease in bone marrow and soft tissues for noninvasive monitoring of high‐risk MM phenotypes. Extramedullary manifestation (EMD) and plasma cell leukemia (PCL) are two aggressive clinical presentations of MM that exhibit high relapse rates. EMD presents with infiltration in other organs, such as lymph nodes, liver, lungs, and central nervous system, while PCL is characterized by free circulation of MM cells in the blood. To achieve this, we generated a disseminated disease model by intravenously injecting luciferase expressing MM1S (Figure  5A) or MOLP8 cells to track cell engraftment in NSG mice. [⁶⁸Ga]Ga‐AJ206 PET showed the distribution of radioactivity throughout the body as anticipated from the disseminated model developed by intravenous injection of MM cells (Figures S9 and S10, Supporting Information). Also, specific accumulation of radioactivity is observed in bone lesions and other organs such as lungs and liver in MM1S (Figure 5B and Figure S8A, Supporting Information) and MOLP8 tumor models (Figure 5D). This finding was further validated by bioluminescence imaging in the MM1S model (Figure 5A) and ex vivo PET imaging in the MOLP8 model (Figure 5D). Furthermore, IHC analysis confirmed high expression of CD38 in mice injected with MM1S‐Luc (Figure 5C) and MOLP8 cells (Figure 5E) in bones as well as in liver and lungs (Figures S9 and S10, Supporting Information). Moreover, analysis of bone lesions from MM1S‐Luc and MOLP8 models showed differential [⁶⁸Ga]Ga‐AJ206 uptake, indicating that different CD38 levels can be differentiated by [⁶⁸Ga]Ga‐AJ206 PET (Figure 5F,G). We also extracted cells from the bone marrow of those mice, which showed high CD38 expression, thus confirming that the observed uptake is indeed CD38 specific (Figure 5H).

Figure 5.

Validation of [⁶⁸Ga]Ga‐AJ206 specificity for CD38 in disseminated MM disease models and primary plasma cell leukemia xenografts. A) IVIS‐bioluminescence image of luciferase expressing MM1S disseminated tumor model. B) In vivo uptake of [⁶⁸Ga]Ga‐AJ206 in lungs and bones in MM1S‐Luc bearing mice. C) Flow cytometry analysis of CD38 expression in lungs harvested from MM1s‐Luc cells injected mice. Lungs from PBS treated mice were used as controls (top panel). IHC staining of bones shows CD38 expression (bottom panel). D) Rendered in vivo and ex vivo [⁶⁸Ga]Ga‐AJ206‐PET images of lower limbs of MOLP8 injected animals. E) IHC images of MOLP8 bone marrow tumor and PBS‐treated animals confirmed CD38 expression. F) Quantification of PET signal (%ID/cc) in the bone marrow of PBS, MM1S‐Luc and MOLP8 injected animals. G) Tumor/muscle (T/M) ratios of PET measures in the bone marrow of PBS, MM1S and MOLP8 injected animals. H) Flow cytometry analysis of CD38 expression in cells extracted from the bone marrow of PBS, MM1S‐Luc and MOLP8 cell injected mice. Data in figure F and G are shown as box and whisker plots showing all data points (n = 7 in PBS, n = 5 in MM1S and n = 8 in MOLP8). Ordinary one‐way ANOVA using multiple comparison test. **, P ≤ 0.01; *** P ≤ 0.001; ****, P ≤ 0.0001.

Extending our studies of [⁶⁸Ga]Ga‐AJ206 to PDXs, we first assessed the CD38 expression on MM cells from two anonymized patient samples and found expression within the range of previous reported expression levels (Figure  6A). We then established primary patient‐derived xenografts (PDXs) and carried out imaging studies. [⁶⁸Ga]Ga‐AJ206 PET showed specific accumulation of radioactivity in both the PDXs that reflected the CD38 expression detected by flow cytometry pre‐ inoculation and by IHC of the tumor sections (Figure 6B,C). Collectively, these results indicate that [⁶⁸Ga]Ga‐AJ206 could aid in detection and monitoring of MM in both soft tissue and bone marrow.

Figure 6.

Validation of [⁶⁸Ga]Ga‐AJ206 specificity for CD38 in primary plasma cell leukemia xenografts. A) Flow cytometry histograms of CD38 expression in primary cells. PDX‐1 is from the peripheral blood of a relapsed/refractory MM patient with secondary plasma cell leukemia and PDX‐2 is from a newly diagnosed MM patient bone marrow. Neither had exposure to anti‐CD38 therapies. B) Static whole‐body PET/CT images of PDX bearing mice at 60 min postinjection of [⁶⁸Ga]Ga‐AJ206. (tumor in dashed red lines) C) IHC analysis of CD38 expression in PDXs.

2.6. Evaluation of Therapy Induced CD38 Pharmacodynamics with [⁶⁸Ga]Ga‐AJ206 in PDX Models

We next evaluated the potential of [⁶⁸Ga]Ga‐AJ206 to visualize therapy induced changes in CD38 expression. MM is often treated with the anti‐CD38 antibody daratumumab, but its response is heterogeneous and linked to CD38 receptor density. Daratumumab treatment can also cause transient downregulation in CD38 expression and heterogenous responses.^{[ 32 ]} As a result, there are efforts underway to improve daratumumab efficacy by increasing CD38 expression using treatments such as ATRA.^{[ 11} , ^{12 ]} In a proof‐of principle study to investigate the potential of [⁶⁸Ga]Ga‐AJ206 to non‐invasively evaluate therapy induced changes in CD38 expression, we first treated four MM cell lines with ATRA and measured changes in CD38 expression. Our results showed that ATRA treatment indeed increased CD38 expression (Figure  7A). Furthermore, when we incubated ATRA treated cells with [⁶⁸Ga]Ga‐AJ206, we observed a significant increase in radioactivity uptake compared to control treatment (Figure 7B). To validate the in vitro results, we performed PET imaging studies in PDX models. A significant increase in radioactivity uptake in tumors in mice were observed following treatment with ATRA compared to pre‐treatment, and confirmed by CD38 IHC of tumor sections taken from vehicle and ATRA treated tumors (Figure 7C,D,G; Figures S12A and S13, Supporting Information). Also, [⁶⁸Ga]Ga‐AJ206 uptake in the PDXs was heterogeneous (Figure 7D–F), which correspond to variable CD38 expression detected by flow cytometry and IHC analysis of those tumors (Figure 7G; Figure S12B,C, Supporting Information). In conclusion, this study provides evidence that [⁶⁸Ga]Ga‐AJ206 has the potential to detect therapy induced changes in CD38 expression in vitro and in vivo, which could be useful in efforts to improve the efficacy of anti‐CD38 therapies.

Figure 7.

In vitro and in vivo detection of ATRA treatment induced changes in CD38 expression by [⁶⁸Ga]Ga‐AJ206 PET in MM cells and PDXs. A) Flow cytometry analysis of ATRA induced changes in surface expression of CD38 in MM cells. B) In vitro uptake of [⁶⁸Ga]Ga‐AJ206 (%IA) in MM cells treated with ATRA or vehicle control. Cells were incubated with [⁶⁸Ga]Ga‐AJ206 at 4 °C for 1 h. C) Static whole‐body PET/CT images of PDX bearing NSG mice before and after treatment with ATRA. Red circles indicate tumor. D) Quantification of PET signal in tumors pre‐ and post‐treatment with ATRA. E) Ratio of PET signal in tumors before (U ₀) and after ATRA (U _t) treatment. F) Tumor/muscle ratio of PET measures before and after ATRA treatment G) CD38 IHC of PDXs of untreated and post‐ATRA treated mice; data in figures A and B are represented as mean ± SD (n = 3 or 4) and significance was calculated by multiple unpaired t test; data in figure D–F are shown for individual mice and significance was calculated using paired t test. ns, P ≥ 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

3. Discussion

In this study, we demonstrated that a new ⁶⁸Ga‐labeled radiotracer, [⁶⁸Ga]Ga‐AJ206, exhibits high affinity for CD38 and optimal pharmacokinetics with excellent image contrast within 60 minutes after administration, aligning with standard clinical workflow and potentially allowing for facile translation into human imaging. Through tumor models, we confirmed that [⁶⁸Ga]Ga‐AJ206 detects varying levels of CD38 expression and differentiates CD38 levels in bone lesions in disseminated disease models. Our pre‐clinical findings indicate that [⁶⁸Ga]Ga‐AJ206 has the potential to be a useful radiotracer in clinical settings for monitoring therapy response and disease progression in MM. Beyond MM, the expression of CD38 in other types of cancer, which is generally more heterogeneous than in MM, suggests that [⁶⁸Ga]Ga‐AJ206 may have a role in selecting those patients that might benefit from CD38 directed therapies. Across all types of CD38 expressing cancers, [⁶⁸Ga]Ga‐AJ206 provides an opportunity to identify new imaging biomarkers for disease prognosis.

Taking advantage of high CD38 expression in MM, several CD38‐targeting therapies have been developed or are in different phases of clinical development.^{[ 8 ]} These include monoclonal antibodies such as daratumumab and isatuximab, which are approved for treating MM, as well as other forms of therapies such as ADCs, T‐cell engagers, and CAR‐T cells.^{[ 8} , ^{33 ]} Responses to anti‐CD38 mAbs has been linked to CD38 receptor density and MM cells with higher levels of CD38 expression are more susceptible to anti‐CD38‐mediated therapeutic effects compared to low CD38 expression levels.^{[ 8} , ¹¹ , ³² , ^{34 ]} Also, there is substantial heterogeneity in response among patients treated with these therapies.^{[ 11} , ³⁵ , ³⁶ , ^{37 ]} For example, daratumumab has been shown to induce durable responses in heavily pretreated patients, but the majority of responding patients eventually develop progressive disease during daratumumab monotherapy. Primary resistance to daratumumab has also been linked to CD38 receptor density.^{[ 38 ]} There is a current focus on enhancing CD38 expression on MM cells in order to capitalize on the wide range of effective treatment options and improve overall efficacy.^{[ 11} , ^{12 ]} Consequently, the precise detection of measurable residual disease will gain greater significance for the expanding patient population, enabling them to benefit from emerging therapeutic alternatives. These clinical observations suggest that real‐time, whole‐body measurements of CD38 expression using molecularly targeted PET radiotracers could provide more accurate information for guiding anti‐CD38 therapies in MM than current single‐time point and single‐lesion flow cytometry measurements. While molecularly targeted imaging agents have proven valuable for diagnosing and guiding treatment in breast, prostate, and various other cancer types,^{[ 39} , ⁴⁰ , ^{41 ]} their potential benefits have yet to be fully realized in the context of MM. Also, MM can be spatially heterogenous, and PET/CT is good at detecting extramedullary disease and focal lesions in the bone marrow. Moreover, bone marrow testing is invasive and a noninvasive methodology that is even a fraction as sensitive could help to prevent the need for bone marrow MRD testing in those who are positive by imaging, and it could be used in conjunction with the bone marrow testing in cases where there are focal lesions.

Considering the importance of CD38 expression in MM, radiolabeled and fluorescence conjugates of daratumumab have been tested in preclinical models and patients.^{[ 13} , ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸ , ¹⁹ , ²⁰ , ^{21 ]} Several clinical trials are ongoing to capture the heterogeneity in CD38 levels in the whole body using radiolabeled analogs of the FDA approved anti‐CD38 antibodies (mAbs). Radiolabeled anti‐CD38 mAb (⁸⁹Zr‐daratumumab) localization to osseous MM sites was visualized by PET with appreciable contrast by day 7.^{[ 19 ]} In another clinical trial using ⁶⁴Cu‐daratumumab, lesions that were ¹⁸F‐FDG negative but positive in CD38‐PET were found to be positive for MM involvement on biopsy,^{[ 42 ]} indicating the potential for CD38‐PET for improved disease burden assessment. In addition, several small protein derived analogs including nanobodies and single domain antibodies have been developed and evaluated in preclinical models of MM.^{[ 22} , ⁴³ , ^{44 ]} Most of those agents provide CD38 specific images within few hours of radiotracer injection. While effective, those agents also show nonspecific uptake in several tissues including liver and lungs and notably high kidney uptake (>100% ID g⁻¹).^{[ 22} , ⁴³ , ^{44 ]} They present low tumor‐to‐muscle ratios, likely attributed to the size and nature of these small proteins. In contrast, our peptide tracers are small (≈2.1 kDa), synthetic, exhibit tractable pharmacokinetics, and take advantage of the inherent sensitivity of PET and the higher contrast resolution characteristics of ⁶⁸Ga. As such, we observed the potential of [⁶⁸Ga]Ga‐AJ206 to detect varying CD38 levels in a variety of tumor models that is corroborated by CD38 IHC. In addition to the high‐contrast images observed with [⁶⁸Ga]Ga‐AJ206, the notable high tumor‐to‐muscle (>25) and tumor‐to‐blood (>7) ratios coupled with low kidney uptake (16%ID g⁻¹) observed at 60 min highlight the benefits of employing small peptides as imaging agents over small proteins. Despite the superior contrast seen with[⁶⁸Ga]Ga‐AJ206, the tumor uptake values are relatively moderate, indicating the need for additional refinement and development of these peptide analogs.

CD38 is expressed at relatively low levels on several hemopoietic cells including regulatory T cells (Tregs) and natural killer (NK) cells, myeloid‐derived suppressor cells (MDSCs) and in normal prostate.^{[ 45 ]} CD38 expression dependent uptake of [⁶⁸Ga]Ga‐AJ206 in those cells and tissues could not be assessed due to the human specificity of the peptide. Nevertheless, our results suggest that CD38 specific low‐molecular‐weight diagnostic agents may have a number of applications in MM, including assessment of total CD38 levels, predicting response to therapy, and determining CD38 occupancy by mAbs, similar to what has been shown by us for PD‐L1 therapeutics.^{[ 25} , ²⁶ , ³¹ , ^{46 ]} Also, clinical trial evaluation of ATRA‐induced changes in CD38 expression was evaluated in peripheral blood samples but not in bone marrow samples due to the invasive nature of the procedure.^{[ 47 ]} The potential of [⁶⁸Ga]Ga‐AJ206 in quantifying the pharmacodynamics of CD38 during therapy strongly suggests that noninvasive monitoring of CD38 expression may find use in how we monitor and assess the effectiveness of various therapies. Moreover, non‐invasive CD38 monitoring is expected to have a broader application across different disease states of MM and may be applicable to a range of malignancies with known CD38 involvement, such as NK/T cell lymphoma, T‐cell acute lymphoblastic leukemia, and primary effusion lymphoma.

4. Conclusions

In summary, we report the development of a first‐in‐class peptide‐based imaging agent, [⁶⁸Ga]Ga‐AJ206, for the detection of CD38 expression using PET. This novel agent, [⁶⁸Ga]Ga‐AJ206, possesses the essential characteristics necessary for clinical application, as it delivers high‐contrast CD38‐specific images across various multiple myeloma models. Furthermore, we show that [⁶⁸Ga]Ga‐AJ206‐PET has the potential to monitor CD38 pharmacodynamics during therapy. This capability could have significant implications for advancing CD38 drug development, as well as guiding the use of approved and novel therapies in MM and other conditions involving CD38.

5. Experimental Section

Chemicals

NOTA‐NHS ester was purchased from CheMatech, and all Fmoc protected amino acids, TIPS, HOBT, and HBTU were obtained from Chem‐impex. Fmoc‐PEG₃‐COOH and ^t Bu₃‐DOTA‐COOH were purchased from Ambeed, while DIPEA, TFA, DODT, TBMB, and DMF were obtained from Sigma‐Aldrich.

Synthesis of AJ205

AJ205 was chemically synthesized using Liberty Blue CEM automatic peptide synthesizer employing Fmoc based solid‐phase peptide synthesis on Rink Amide resin in a 0.1 mmol scale (Scheme S1, Supporting Information). Coupling reaction was carried out using Oxyma (0.5 mmol), DIC (1 mmol), and Fmoc‐AA‐OH (0.5 mmol) in DMF with microwave assisted reaction for 2 min. Fmoc group was deprotected using 20% piperidine in DMF (3 mL) for 1 min with microwave assistance. Next, PEG linker was incorporated by using HBTU (0.5 mmol), HOBT (0.5 mmol), DIPEA (0.5 mmol) and Fmoc‐NH‐PEG₃‐COOH (0.5 mmol) in DMF at room temperature for 1.5 h and then Fmoc group was deprotected using 20% piperidine (4 mL) in DMF for 40 min at room temperature. Once the sequence was completed on the resin, the peptidyl‐resin was treated with 4 mL of cleavage cocktail (TFA:TIPS:DODT:H₂O; 92.5:2.5:2.5:2.5) for 4 h at room temperature. The cleaved reaction mixture was precipitated with diethyl ether to obtain the linear peptide as a white solid.

For the cyclization, the linear peptide (98 mg, 0.06 mmol) was dissolved in 100 mL of water:acetonitrile (1:1) and treated with an aqueous solution of tris(2‐carboxyethyl)phosphine (TCEP) (115 mg, 0.4 mmol) followed by Et₃N (400 µL, 2.9 mmol). 1,3,5‐Tris(bromomethyl)benzene (TBMB) (34 mg, 0.1 mmol) was dissolved in 2 mL of acetonitrile and added slowly to the reaction mixture over an hour. The reaction mixture was stirred at room temperature for 24 h and quenched with TFA. The volatiles were removed, and the crude mixture was purified on a reversed‐phase high‐performance liquid chromatography (RP‐HPLC) system using a preparative C‐18 Phenomenex column (5 mm, 21.5 × 250 mm Phenomenex, Torrance, CA). The HPLC condition was gradient elution starting with 10% acetonitrile: water (0.1% TFA) and reaching 60% acetonitrile: water (0.1% TFA) in 40 min at a flow rate of 8 mL min⁻¹. The product AJ205 was collected at retention time (RT) ≈28.2 min. Acetonitrile was evaporated under reduced pressure and lyophilized to form an off‐white powder with a 37% yield (40 mg). The sequence of the AJ205 peptide is NH₂‐PEG₃‐ACVPCADFPIWYC‐NH₂ with TBMB‐based cyclization. This peptide was characterized by MALDI‐TOF‐MS. The theoretical chemical formula is C₈₇H₁₁₈N₁₆O₂₀S₃ with an exact mass of 1802.79 and a molecular weight of 1804.17. The theoretical MALDI‐TOF‐MS mass [M + H]⁺ was 1803.8; the observed ESI‐MS mass [M + H]⁺ was 1804.0.

Synthesis of AJ206

To a stirred solution of AJ205 (2.1 mg, 1.2 µmoles) in 200 µL of DMF in a reaction vial, NOTA‐NHS ester (2.0 mg, 2.8 µmoles) and DIPEA (5 µL, 25 µmoles) were added and stirred at room temperature for 3 h (Scheme S2, Supporting Information). DMF was evaporated using a rotary evaporator under reduced pressure and the residual product was purified on a RP‐HPLC system using a semipreparative C‐18 Luna column (5 mm, 10 × 250 mm Phenomenex, Torrance, CA). The HPLC condition was gradient elution started with 5% acetonitrile:water (0.1% TFA) and reached at 95% acetonitrile: water (0.1% TFA) in 20 min at a flow rate of 5 mL min⁻¹. The product AJ206 was collected at RT ≈11.8 min. Acetonitrile was evaporated under reduced pressure and lyophilized to form an off‐white powder with a 52% yield, which was characterized by MALDI‐TOF‐MS. The theoretical chemical formula of AJ206 is C₉₉H₁₃₇N₁₉O₂₅S₃ with an exact mass of 2087.92 and molecular weight of 2089.47. The theoretical MALDI‐TOF‐MS mass [M + H]⁺ is 2088.9, and the observed ESI‐MS mass [M + H]⁺ is 2089.1.

Affinity Measurements by Surface Plasmon Resonance (SPR)

The affinity of AJ206 for hCD38 and mCD38 recombinant proteins were evaluated by SPR. The experiments were conducted using a Biacore T200 instrument with a CM5 chip at 25 °C. The ligands used were His‐Tagged human CD38 (R&D systems, catalog # 2404‐AC, 43 kDa, 0.5 mg mL⁻¹ stock concentration) and mouse CD38 proteins (R&D systems, catalog # 4947‐AC, 40 kDa, 1.81 mg mL⁻¹ stock concentration), which were immobilized onto the CM5 chip. AJ206 (2089.2 Da, 10 × 10⁻³ m stock concentration) was used as the analyte, which flowed over the ligand immobilized surface. FC2 was used as the experimental flow cell, while FC1 served as the reference. Anti‐His antibody (1 mg mL⁻¹ stock concentration) was immobilized on both FC1 and FC2 using standard amine coupling chemistry. The immobilization running buffer used was PBS‐P (20 × 10⁻³ m phosphate buffer pH 7.4, 137 × 10⁻³ m NaCl, 2.7 × 10⁻³ m KCl, 0.05% v/v surfactant P20). Human CD38 was captured onto FC2 at a level of ≈860 RU, with a 1:20 dilution and 25 µg mL⁻¹ diluted concentration in PBS‐P. Mouse CD38 was captured onto FC4 at a level of 1360 RU, with a 1:50 dilution and 36.2 µg mL⁻¹ diluted concentration in PBS‐P. The theoretical R _max values were calculated based on the captured response values and are presented in Table  2 , assuming a 1:1 interaction mechanism. Overnight kinetics were performed for all analytes in the presence of PBS‐P+1% DMSO. The flow rate of all analyte solutions was maintained at 50 µL min⁻¹. The contact and dissociation times used were 120 and 360 s, respectively. Surface regeneration was achieved by injecting glycine pH 1.5 for 20 s, which takes away all captured ligands onto FC2. Fresh ligands were captured at the beginning of each injection cycle. The analyte concentrations injected ranged from 300 × 10⁻⁹ m down to 1.2 × 10⁻⁹ m with threefold serial dilutions, and all analytes were injected in triplicate.

Table 2.

Parameters of affinity measurements of AJ206 by SPR.

Ligand	FC	Analyte	Ligand binding (RU)	MWL [Da]	MWA [Da]	Stochiometric ratio	R max
Human CD38	2	AJ206	860	40 000	2089.6	1:1	39.2
Mouse CD38	4	AJ206	1360	40 000	2089.6	1:1	71.0

Synthesis of [^natGa]Ga‐AJ206

The synthesis of [^natGa]Ga‐AJ206 was carried out by adding 10 µL of aqueous 0.1 m [^natGa]GaCl₃ solution and 0.6 mL of 0.1 m HCl to a stirred solution of AJ206 (0.1 mg, 0.05 µmoles) in 200 µL of 1 m NaOAc buffer (pH 5.0) in a reaction vial. The reaction mixture was incubated at 65 °C for 30 min and then purified on a RP‐HPLC system using a semipreparative C‐18 Luna column (5 mm, 10 × 250 mm Phenomenex, Torrance, CA). The HPLC gradient elution condition started with 5% acetonitrile: water (0.1% TFA) and reached at 95% acetonitrile: water (0.1% TFA) in 20 min at a flow rate of 5 mL min⁻¹. The product [^natGa]Ga‐AJ206 was collected at RT ≈11.8 min. The acetonitrile was evaporated under reduced pressure and lyophilized to form an off‐white powder, which was characterized by MALDI‐TOF‐MS. The theoretical chemical formula is C₉₉H₁₃₄GaN₁₉O₂₅S₃ with an exact mass of 2153.82 and a molecular weight of 2156.17. The theoretical MALDI‐TOF‐MS mass [M + H]⁺ was 2154.8, and the observed ESI‐MS mass [M + H]⁺ was 2154.1.

Synthesis of [⁶⁸Ga]Ga‐AJ206

The ⁶⁸Ge/⁶⁸Ga generator was manually eluted using 6 mL of 0.1 m HCl (Ultrapure trace‐metal‐free) in four different fractions (2.4, 1, 1, and 1.4 mL). To a microcentrifuge vial (1.5 mL) containing 200 µL of 1 m NaOAc buffer (pH = 5) and 20 µg of AJ206 (10 nmoles), 3‐4 mCi of [⁶⁸Ga]GaCl₃ in 0.6 mL from the second fraction was added (Scheme S3, Supporting Information). The reaction mixture was incubated for 12 min at 65 °C in a temperature‐controlled heating block and purified on a RP‐HPLC system using a semi‐preparative C‐18 Luna column (5 mm, 10 × 250 mm Phenomenex, Torrance, CA). The HPLC condition was gradient elution starting with 5% acetonitrile: water (0.1% TFA) and reaching 95% acetonitrile: water (0.1% TFA) in 20 min at a flow rate of 5 mL min⁻¹. The radiolabeled product [⁶⁸Ga]Ga‐AJ206 was collected at RT ≈11.9 min, with decay‐corrected radiochemical yield of 92 ± 10.5% (n = 35). The desired radiolabeled fraction was concentrated under a stream of N₂ at 60 °C, formulated in 10% EtOH in saline, and used for in vitro and in vivo studies. The whole radiolabeling process was completed in approximately 35 min. Quality control, stability studies, and chemical identity were also performed on the same HPLC system using the same HPLC gradient as described above.

Determination of Partition Coefficient

Partition coefficients, logD (pH7.4) value, were determined according to a literature procedure.^{[ 46 ]} Briefly, a 10 µL solution of [⁶⁸Ga]Ga‐AJ206 (around 10 µCi) was added to a solution of 1‐octanol (200 µL) mixed with phosphate buffered saline (PBS) (190 µL) in a 1.5 mL centrifuge tube. After the mixture was vigorously shaken and vortexed, it was centrifuged at 3000 rpm for 5 min. Aliquots (10 µL) were removed from the two phases, and the radioactivity was measured on an automated gamma counter (1282 Compugamma CS, Pharmacia/LKB Nuclear, Inc., Gaithersburg, MD). The logD was calculated as the average log ratio value of the radioactivity in the 1‐octanol fraction and the PBS fraction from the four samples.

Cell Culture

MOLP8 cells were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Germany, under the material transfer agreement guidelines with Johns Hopkins University. Other cell lines (U266, RPMI8226, MM1S and MM1S‐Luc) were gifted by a collaborator. Prior to the start of the experiments, all cell lines were authenticated by STR profiling at the Johns Hopkins Genetic Resources Facility. Routine mycoplasma testing was conducted, and the cell lines were passaged for no more than 3 months. New cultures were initiated from vials of frozen cells. All cell lines (U266, RMPI8226, MM1S, MM1S‐Luc, and MOLP8) were cultured in the recommended media and maintained in an incubator at 37 °C in an atmosphere containing 5% CO₂. All cells were supplemented with fetal bovine serum, 1% P/S antibiotics, and 1 × 10⁻³ M ‐m L‐Glutamine.

In Vitro Cellular Binding Assays

In vitro binding assays were conducted to determine the binding of [⁶⁸Ga]Ga‐AJ206 to U266, RPMI8226, MM1S, and MOLP8 cells. Approximately 1 µCi of [⁶⁸Ga]Ga‐AJ206 was incubated with 1 × 10⁶ cells in 100 µL of culture media for 60 min at 4 °C. After incubation, cells were washed three times with ice‐cold PBS containing 0.1% tween‐20 and counted on an automated gamma counter (1282 Compugamma CS, Pharmacia/LKB Nuclear, Inc., Gaithersburg, MD). Blocking was performed with 2 × 10⁻⁶ m of AJ206 to demonstrate CD38‐specific cellular binding of [⁶⁸Ga]Ga‐AJ206. All cellular uptake studies were performed in quadruplicate for each cell line and repeated three times.

Internalization Assay

Approximately 5 µCi of [⁶⁸Ga]Ga‐AJ206 was incubated with 1 × 10⁶ RPMI8226 cells in 100 µL of culture media at 37 °C in quadruplet for each group. After incubation, cells were washed three times with ice‐cold PBS containing 0.1% tween‐20 at predetermined time‐points (3, 10, 30, 60 and 120 min). To remove the receptor‐bound radiotracer, an acid wash was carried out twice with a 0.1 m glycine buffer pH 3.0 for 5 min on ice and this wash was collected in different tubes to obtain the receptor bound fraction. Finally, radioactivity was measured in cell pellets and acid wash using gamma counter to obtain total cell bound and internalized radiotracer.

Flow Cytometry Analysis of CD38 Expression

To evaluate CD38 surface expression, cells (1 × 10⁶) were stained with PE‐labelled anti‐human CD38 monoclonal antibody (clone HB‐7, BioLegend Cat # 356604) in FACS buffer (0.5% FBS with 2 mm EDTA) for 30 min on ice. After washing and resuspending in FACS buffer, data were acquired on a BD Accuri C6 plus flow cytometer and analyzed using FlowJo software. Receptor density measurements were performed using Quantibrite Beads (BD Biosciences, cat #340495), which contain four levels of phycoerythrin (PE) per bead. Gates were drawn on low, medium low, medium high, and high PE binding beads, and the geometric mean from these populations was correlated with the lot‐specific PE molecule/bead on a logarithmic scale. This correlation was used to translate cell population geometric mean to receptor/cell for the respective cell type.

Western Blotting

To assess CD38 protein expression in MM cells, lysed cells were denatured using Laemmli SDS sample buffer with beta‐mercaptaethanol, loaded onto SDS‐PAGE gels, and transferred to PVDF membranes. Blots were probed with rabbit monoclonal anti‐human CD38 antibody (Clone RM388, Thermo Scientific, cat# MA5‐36061) after blocking with 5% BSA. GAPDH was used as a loading control (Rabbit polyclonal, Clone D16H11, CST, cat# 5174). Blots were incubated overnight at 4 °C, washed, and incubated with anti‐rabbit IgG, HRP‐linked secondary antibody (CST, cat #7074) to detect binding. Immunoreactivity was detected using super signal west Pico plus Chemiluminescent substrate (Thermo Scientific) and analyzed on Imaging System using Chemidoc scanner.

Tumor Models

Animal studies were performed under Johns Hopkins University Animal Care and Use Committee (ACUC‐approved protocol (Principal investigator: Sridhar Nimmagadda, Ph.D. and Protocol number M021M175). Male and female NSG mice (5‐6 weeks old) were used to establish xenografts by administering 3 million cells (50% Corning Matrigel) subcutaneously to form various tumor models within 3–4 weeks. Imaging or biodistribution studies (n = 3–5) were conducted on mice with tumor volumes of 100–200 mm³. Disseminated tumor models were developed by injecting 4–5 million cells in saline intravenously for MOLP8 and MM1S‐Luc models (n = 4–5). Luciferase expressing MM1S cell line (MM1S‐Luc) was used to monitor cancer cell dissemination in the body. Tumor establishment was confirmed by injecting D‐Luciferin intraperitoneally (150 mg kg⁻¹) and imaged mice after 10 min in IVIS bioluminescence imaging system. Bone marrow tumors were ready for imaging after 25–30 d of implantation.

To establish PDX models, immune‐deficient NSG male mice (NOD/Shi‐scid IL‐2rgnull), aged 6–8 weeks, were employed after being irradiated at an absorbed dose of 200 cGy. PCL cells, sourced from two patients and anonymized, were obtained from Dr. Gocke's laboratory. Under sterile conditions, the cells were thawed and treated with DNAse at a concentration of 20 µg mL⁻¹. After cell counting, 30 000 cells were subjected to flow cytometry analysis to assess CD38 expression. The remaining cells were subcutaneously inoculated at a concentration of 5 × 10⁶ cells in 100 µL of a solution comprising 60% Matrigel Basement Matrix (Corning 356230) diluted with HBSS. The animals were included in the experiments once the PDX volumes reached tumor volumes of 100–200 mm³.

Bone marrow and peripheral blood samples were collected from MM patients or healthy donors who gave informed consent, in compliance with the Declaration of Helsinki. This was approved by the Institutional Review Board at the Johns Hopkins Medical Institutes. The mononuclear cells were then isolated via density centrifugation using Ficoll‐Paque, a product of Pharmacia, located in Piscataway, NJ.

Evaluation of Pharmacokinetics of [⁶⁸Ga]Ga‐AJ206

Dynamic PET images were acquired on a Simultaneous 7T Bruker PET‐MR scanner to evaluate the pharmacokinetics of [⁶⁸Ga]Ga‐AJ206. Mice bearing MOLP8 tumor xenografts were anesthetized under 2.5% isoflurane and a catheter was fixed in the tail vein before being secured on the PET‐MR bed. An activity of ≈250 µCi (9.3 MBq) of [⁶⁸Ga]Ga‐AJ206 was administered intravenously and whole‐body PET dynamic scans were performed starting from −1 to 5 min in 30 s intervals, followed by scans at 5–15 min in 1 min intervals, 15_–30 min in 3 minute intervals, 30–60 min in 5 min intervals, and 60–90 min in 10 min intervals. The acquired PET data were reconstructed and corrected for radioactive decay and dead time using ParaVision 360 V2 by Bruker. The percentage of injected dose per cc (%ID/cc) values were obtained by drawing ROI on the tumor, muscle, heart, liver and kidney using PMOD software, and image fusion and visualization were also performed using PMOD software.

PET‐CT Imaging of Mouse Xenografts

Mice bearing flank tumors and disseminated disease were injected intravenously with [⁶⁸Ga]Ga‐AJ206 and PET images were acquired 60 min after injection of the radiotracer. Images were acquired in 2 bed positions for a total of 10 min using an ARGUS small‐animal PET/CT scanner. Images were reconstructed using 2D‐OSEM and corrected for radioactive decay and dead time. Image fusion, visualization, and 3D rendering were accomplished using Amira 2020.3.1. PET images were quantified by drawing ROI on tissues using Amide 1.0.6 and reported as %ID/cc.

Ex Vivo Biodistribution

Mice with 100–200 mm³ tumor volume were used for ex vivo biodistribution studies. For radiotracer pharmacokinetics studies, mice with MOLP8 tumor xenografts received ≈80 µCi (2.96 MBq) [⁶⁸Ga]Ga‐AJ206 and were sacrificed at pre‐determined time points (5, 30, 60 and 120 min). Similarly, for dosimetry study, mice with MM1S tumor xenografts received ≈80 µCi (2.96 MBq) of [⁶⁸Ga]Ga‐AJ206 and were sacrificed at pre‐determined time points (5, 30, 60, 90 and 120 min). All other mice were injected with ≈50µCi (1.85 MBq) [⁶⁸Ga]Ga‐AJ206 and were sacrificed at 60 min. The selected tissues were collected, weighed, counted, and their %ID g⁻¹ values calculated for biodistribution analysis. The tissues included blood, muscle, tumor, thymus, heart, lung, liver, pancreas, stomach, small intestine, large intestine, spleen, adrenals, kidney, bladder, femur, and brain.

Harvesting of Tissues for Immunohistochemistry and Flow Cytometry

After the imaging study, mice were humanely euthanized and their tissues Including inflated lungs, and tumors were fixed in 4% paraformaldehyde and sent to oncology tissue services (OTS) at JHU. Tissue sections of 4 µm thickness were prepared from paraffin‐embedded tissues. Bones that were harvested were fixed and decalcified for 15 d in 10% EDTA on a slow‐speed rocking shaker.

For flow cytometric analysis, single‐cell suspensions were prepared from bone marrow and lungs. Femurs were properly blenched and cut from the epiphysis, and cells were collected directly in tubes. Lungs were chopped and digested in a buffer containing collagenase and DNase, incubated at 37 °C for 30 min, and strained through a 40um cell strainer. Cell pellets were lysed in RBC lysis buffer, and live/dead staining was conducted using near infra‐red 780 dye and CD38 staining, as previously described.

ATRA Treatment Studies

To evaluate the pharmacodynamic effects of ATRA therapy on MM cells, U266, RPMI8226, MM1S, and MOLP8 cells were seeded at a concentration of 0.5 million in 500 µL per well in triplicates in a six‐well plate. A 200 × 10⁻⁹ m working solution of all‐trans retinoic acid (ATRA, Sigma‐Aldrich #554720) was prepared by diluting in culture media. Next, 500 µL of the working solution was added to each well, and the cells were incubated for 48 h at 37 °C in a 5% CO₂ atmosphere. After incubation, cells were transferred to FACS tubes, medium was removed by centrifugation, and the cells were resuspended in FACS buffer for flow cytometry and in vitro binding studies, following the same protocol as previously described.

To assess the pharmacodynamics of ATRA‐induced CD38 expression in vivo, mice bearing PDX tumors (n = 6) were subjected to image using [⁶⁸Ga]Ga‐AJ206 to quantify CD38 expression prior to treatment. Following the pretreatment imaging, the mice were orally administered 500 µg of ATRA per day, prepared in a solution of 1% methyl cellulose, for a duration of 3 d. PET images were acquired on the fourth day after the start of treatment. Subsequently, the mice were humanely euthanized, and their tumors were collected in RPMI media for flow cytometry analysis and in formalin solution for IHC. All images were analyzed using AMIDE software with standard ROI analysis techniques.

Immunohistochemistry

To perform immunohistochemistry, the tissue slides were deparaffinized by baking at 60 °C and washing with xylene and alcohol. Antigen retrieval was carried out using citrate buffer (pH 6.0, 95–100 °C, 20 min) and the endogenous peroxidase and alkaline phosphatase activity was blocked using BioXALL. The primary anti‐human CD38 antibody was applied at a dilution of 1:250 and incubated overnight at 4 °C (Thermo Scientific clone RM388 Cat# MA5‐36061). After washing with PBS, the secondary antibody, Signalstain Boost IHC Detection Reagent (HRP), was applied and incubated for 30 min at room temperature. The slides were washed and developed using ImmPACT DAB substrate (Vector Lab #SK4105). After washing, the slides were counterstained with Mayer's hematoxylin for 1 min, dehydrated using alcohol and xylene, and then cover slipped.

Statistical Analysis

All statistical analyses were performed using Prism 9.0 Software (GraphPad Software, La Jolla, CA). Unpaired Student's t test and one‐ or two‐way ANOVA were utilized for column, multiple column, and grouped analyses, respectively. Paired student t test was used to analyze ATRA treatment studies. Statistical significance was set at ns, P ≥ 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Correlation was performed using simple linear regression without keeping the term constant at zero.

Conflict of Interest

The authors declare no conflict of interest related to this work.

Author Contributions

A.K.S. and K.G. contributed equally to this work. A.K.S.: Conceptualization, methodology, data curation, formal analysis, writing‐original draft, writing‐review and editing. K.G.: Conceptualization, methodology, data curation, formal analysis, writing‐original draft, writing‐review and editing. A.M.: Methodology, data curation, writing‐review and editing G.L.: Data curation, writing‐review and editing I.M.: Data curation, writing‐review and editing. D.K.: Data curation, writing‐review and editing. G.G.: Methodology, formal analysis. P.I.: Formal analysis, writing‐review and editing. S.P.R.: Formal analysis, Writing‐review and editing. R.F.H.: Formal analysis, writing‐review and editing. C.B.G.: Methodology, writing‐review and editing. S.N.: Funding acquisition, conceptualization, methodology, data curation, formal analysis, writing‐original draft, writing‐review and editing.

Supporting information

Supporting Information

Sharma A. K., Gupta K., Mishra A., Lofland G., Marsh I., Kumar D., Ghiaur G., Imus P., Rowe S. P., Hobbs R. F., Gocke C. B., Nimmagadda S., CD38‐Specific Gallium‐68 Labeled Peptide Radiotracer Enables Pharmacodynamic Monitoring in Multiple Myeloma with PET. Adv. Sci. 2024, 11, 2308617. 10.1002/advs.202308617

Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article.