First-in-man study of the PSMA Minibody IR800-IAB2M for molecularly targeted intraoperative fluorescence guidance during radical prostatectomy.

PURPOSE: Prostate-specific membrane antigen (PSMA) is increasingly used to image prostate cancer in clinical practice. We sought to develop and test a humanised PSMA minibody IAB2M conjugated to the fluorophore IRDye 800CW-NHS ester in men undergoing robot-assisted laparoscopic radical prostatectomy (RARP) to image prostate cancer cells during surgery. METHODS: The minibody was evaluated pre-clinically using PSMA positive/negative xenograft models, following which 23 men undergoing RARP between 2018 and 2020 received between 2.5 mg and 20 mg of IR800-IAB2M intravenously, at intervals between 24 h and 17 days prior to surgery. At every step of the procedure, the prostate, pelvic lymph node chains and extra-prostatic surrounding tissue were imaged with a dual Near-infrared (NIR) and white light optical platform for fluorescence in vivo and ex vivo. Histopathological evaluation of intraoperative and postoperative microscopic fluorescence imaging was undertaken for verification. RESULTS: Twenty-three patients were evaluated to optimise both the dose of the reagent and the interval between injection and surgery and secure the best possible specificity of fluorescence images. Six cases are presented in detail as exemplars. Overall sensitivity and specificity in detecting non-lymph-node extra-prostatic cancer tissue were 100% and 65%, and 64% and 64% respectively for lymph node positivity. There were no side-effects associated with administration of the reagent. CONCLUSION: Intraoperative imaging of prostate cancer tissue is feasible and safe using IR800-IAB2M. Further evaluation is underway to assess the benefit of using the technique in improving completion of surgical excision during RARP. REGISTRATION: ISCRCTN10046036: https://www.isrctn.com/ISRCTN10046036 .

Boronotyrosine, a Borylated Amino Acid Mimetic with Enhanced Solubility, Tumor Boron Delivery, and Retention for the Re-emerging Boron Neutron Capture Therapy Field.

Boron neutron capture therapy (BNCT) is a re-emerging binary cellular level cancer intervention that occurs through the interaction of a cancer-specific 10boron (10B) drug and neutrons. We created a new 10B drug, 3-borono-l-tyrosine (BTS), that improves on the characteristics of the main historical BNCT drug 4-borono-l-phenylalanine (BPA). BTS has up to 4 times greater uptake in vitro than BPA and increased cellular retention. Like BPA, BTS uptake is mediated by the l-type amino acid transporter-1 (LAT1) but is less sensitive to natural amino acid competition. BTS can be formulated and bolus dosed at much higher levels than BPA, resulting in 2-3 times greater boron delivery in vivo. Fast blood clearance and greater tumor boron delivery result in superior tumor-to-blood ratios. BTS boron delivery appears to correlate with LAT1 expression. BTS is a promising boron delivery drug that has the potential to improve modern BNCT interventions.

The PET-Tracer 89Zr-Df-IAB22M2C Enables Monitoring of Intratumoral CD8 T-cell Infiltrates in Tumor-Bearing Humanized Mice after T-cell Bispecific Antibody Treatment.

CD8-expressing T cells are the main effector cells in cancer immunotherapy. Treatment-induced changes in intratumoral CD8+ T cells may represent a biomarker to identify patients responding to cancer immunotherapy. Here, we have used a 89Zr-radiolabeled human CD8-specific minibody (89Zr-Df-IAB22M2C) to monitor CD8+ T-cell tumor infiltrates by PET. The ability of this tracer to quantify CD8+ T-cell tumor infiltrates was evaluated in preclinical studies following single-agent treatment with FOLR1-T-cell bispecific (TCB) antibody and combination therapy of CEA-TCB (RG7802) and CEA-targeted 4-1BB agonist CEA-4-1BBL. In vitro cytotoxicity assays with peripheral blood mononuclear cells and CEA-expressing MKN-45 gastric or FOLR1-expressing HeLa cervical cancer cells confirmed noninterference of the anti-CD8-PET-tracer with the mode of action of CEA-TCB/CEA-4-1BBL and FOLR1-TCB at relevant doses. In vivo, the extent of tumor regression induced by combination treatment with CEA-TCB/CEA-4-1BBL in MKN-45 tumor-bearing humanized mice correlated with intratumoral CD8+ T-cell infiltration. This was detectable by 89Zr-IAB22M2C-PET and γ-counting. Similarly, single-agent treatment with FOLR1-TCB induced strong CD8+ T-cell infiltration in HeLa tumors, where 89Zr-Df-IAB22M2C again was able to detect CD8 tumor infiltrates. CD8-IHC confirmed the PET imaging results. Taken together, the anti-CD8-minibody 89Zr-Df-IAB22M2C revealed a high sensitivity for the detection of intratumoral CD8+ T-cell infiltrates upon either single or combination treatment with TCB antibody-based fusion proteins. These results provide further evidence that the anti-CD8 tracer, which is currently in clinical phase II, is a promising monitoring tool for intratumoral CD8+ T cells in patients treated with cancer immunotherapy. SIGNIFICANCE: Monitoring the pharmacodynamic activity of cancer immunotherapy with novel molecular imaging tools such as 89Zr-Df-IAB22M2C for PET imaging is of prime importance to identify patients responding early to cancer immunotherapy.

Cerenkov Luminescence Imaging as a Modality to Evaluate Antibody-Based PET Radiotracers.

Antibodies, and engineered antibody fragments, labeled with radioisotopes are being developed as radiotracers for the detection and phenotyping of diseases such as cancer. The development of antibody-based radiotracers requires extensive characterization of their in vitro and in vivo properties, including their ability to target tumors in an antigen-selective manner. In this study, we investigated the use of Cerenkov luminescence imaging (CLI) as compared with PET as a modality for evaluating the in vivo behavior of antibody-based radiotracers. METHODS: The anti-prostate-specific membrane antigen (PSMA) huJ591 antibody (IgG; 150 kDa) and its minibody (Mb; 80 kDa) format were functionalized with the chelator 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA) and radiolabeled with the positron-emitting radionuclide 64Cu (half-life, 12.7 h). Immunoreactive preparations of the radiolabeled antibodies were injected into NCr nu/nu mice harboring PSMA-positive CWR22Rv1 and PSMA-negative PC-3 tumor xenografts. Tumor targeting was evaluated by both PET and CLI. RESULTS: 64Cu-NODAGA-PSMA-IgG and 64Cu-NODAGA-PSMA-Mb retained the ability to bind cell surface PSMA, and both radiotracers exhibited selective uptake into PSMA-positive tumors. Under the experimental conditions used, PSMA-selective uptake of 64Cu-NODAGA-PSMA-IgG and 64Cu-NODAGA-PSMA-Mb was observed by CLI as early as 3 h after injection, with tumor-to-background ratios peaking at 24 (IgG) and 16 (Mb) h after injection. Targeting data generated by CLI correlated with that generated by PET and necropsy. CONCLUSION: CLI provided a rapid and simple assessment of the targeting specificity and pharmacokinetics of the antibody-based PET radiotracers that correlated well with the behavior observed by standard PET imaging. Moreover, CLI provided clear discrimination between uptake kinetics of an intact IgG and its small-molecular-weight derivative Mb. These data support the use of CLI for the evaluation of radiotracer performance.

A DLL3-targeted antibody-drug conjugate eradicates high-grade pulmonary neuroendocrine tumor-initiating cells in vivo.

The high-grade pulmonary neuroendocrine tumors, small cell lung cancer (SCLC) and large cell neuroendocrine carcinoma (LCNEC), remain among the most deadly malignancies. Therapies that effectively target and kill tumor-initiating cells (TICs) in these cancers should translate to improved patient survival. Patient-derived xenograft (PDX) tumors serve as excellent models to study tumor biology and characterize TICs. Increased expression of delta-like 3 (DLL3) was discovered in SCLC and LCNEC PDX tumors and confirmed in primary SCLC and LCNEC tumors. DLL3 protein is expressed on the surface of tumor cells but not in normal adult tissues. A DLL3-targeted antibody-drug conjugate (ADC), SC16LD6.5, comprised of a humanized anti-DLL3 monoclonal antibody conjugated to a DNA-damaging pyrrolobenzodiazepine (PBD) dimer toxin, induced durable tumor regression in vivo across multiple PDX models. Serial transplantation experiments executed with limiting dilutions of cells provided functional evidence confirming that the lack of tumor recurrence after SC16LD6.5 exposure resulted from effective targeting of DLL3-expressing TICs. In vivo efficacy correlated with DLL3 expression, and responses were observed in PDX models initiated from patients with both limited and extensive-stage disease and were independent of their sensitivity to standard-of-care chemotherapy regimens. SC16LD6.5 effectively targets and eradicates DLL3-expressing TICs in SCLC and LCNEC PDX tumors and is a promising first-in-class ADC for the treatment of high-grade pulmonary neuroendocrine tumors.

Pharmacologically enhanced expression of GPNMB increases the sensitivity of melanoma cells to the CR011-vcMMAE antibody-drug conjugate.

GPNMB is a melanoma-associated glycoprotein that is targeted by the CR011-vcMMAE antibody-drug conjugate (ADC). Previous studies have shown that CR011-vcMMAE induces the apoptosis of GPNMB-expressing tumor cells in vitro and tumor regression in xenograft models. This ADC is currently in clinical trials for melanoma. In the present investigation, a variety of compounds were examined for their ability to increase the expression of GPNMB by cancer cells. These experiments lead to the identification of three distinct groups of compounds that increased GPNMB, some of which were shown to enhance the sensitivity of melanoma cells to CR011-vcMMAE. These data indicate that it may be possible to increase the anticancer activity of CR011-vcMMAE through pharmacological enhancement of GPNMB expression for potential therapeutic benefit.

Treatment parameters modulating regression of human melanoma xenografts by an antibody-drug conjugate (CR011-vcMMAE) targeting GPNMB.

PURPOSE: To investigate the pharmacological properties of the CR011-vcMMAE fully human antibody-drug conjugate (ADC), such as dose titrations, quantitation of the time (days) to complete regression, pharmacokinetics, and schedule dependency. Our prior study characterized a fully human antibody to GPNMB covalently linked to monomethylauristatin E, CR011-vcMMAE, and further demonstrated cell surface staining of melanoma lines susceptible to the immunoconjugate's cytotoxicity (Clin Cancer Res 2005; 12(4): 1373-1382). METHODS: The human SK-MEL-2 and SK-MEL-5 melanoma xenografts were used in athymic mice to assess anti-tumor efficacy. After s.c. implantation, tumors became established (60-100 mg), and treatment commenced by i.v. injection of the immunoconjugate or vinblastine or paclitaxel. Short-term anti-tumor effects (inhibition of tumor growth) and long-term effects (complete regression) were observed. RESULTS: CR011-vcMMAE induced regression of established human SK-MEL-2 and SK-MEL-5 xenografts at doses from 1.25 to 80 mg/kg treatment when administered intravenously every 4 days (4 treatments); strikingly, regressions were not associated with re-growth during the observation period (200 days). The disappearance rate of implants was dose dependent (minimum time, 18.5 days). Detectable serum CR011-vcMMAE >or=1 microg/mL (approximately 0.01 microM) was observed for >30 days post-dose; CR011-vcMMAE showed an elimination half-life of 10.3 days. A low volume of distribution suggested that CR011-vcMMAE was confined to blood and interstitial fluid. CR011-vcMMAE could be delivered by either a single bolus dose or by intermittent dosing (i.e., every 1, 2, 4, 8, or 16 days) with no discernible differences in the proportion of tumor-free survivors, indicating a lack of schedule dependency. The antibody-drug conjugate produced complete regressions, but the equivalent doses of free monomethylauristatin E or unconjugated antibody did not show anti-tumor effects. In addition, decreases in plasma tumor-derived human interleukin-8 coincided with tumor nodule disappearance. CONCLUSIONS: Short-term anti-tumor effects and long-term effects (complete regression) were observed with CR011-vcMMAE, but not with the reference agents. These results suggest that CR011-vcMMAE may provide therapeutic benefit in malignant melanoma.

Potent cytotoxicity of an auristatin-containing antibody-drug conjugate targeting melanoma cells expressing melanotransferrin/p97.

Identifying factors that determine the sensitivity or resistance of cancer cells to cytotoxicity by antibody-drug conjugates is essential in the development of such conjugates for therapy. Here the monoclonal antibody L49 is used to target melanotransferrin, a glycosylphosphatidylinositol-anchored glycoprotein first identified as p97, a cell-surface marker in melanomas. L49 was conjugated via a proteolytically cleavable valine-citrulline linker to the antimitotic drug, monomethylauristatin F (vcMMAF). Effective drug release from L49-vcMMAF likely requires cellular proteases most commonly located in endosomes and lysosomes. Melanoma cell lines with the highest surface p97 expression (80,000-280,000 sites per cell) were sensitive to L49-vcMMAF whereas most other cancer cell lines with lower p97 expression were resistant, as were normal cells with low copy numbers (< or = 20,000 sites per cell). Cell line sensitivity to L49-vcMMAF was found by immunofluorescence microscopy to correlate with intracellular fate of the conjugate. Specifically, L49-vcMMAF colocalized with the lysosomal marker CD107a within sensitive cell lines such as SK-MEL-5 and A2058. In contrast, in resistant cells expressing lower p97 levels (H3677; 72,000 sites per cell), L49-vcMMAF colocalized with caveolin-1, a protein prominent in caveolae, but not with CD107a. Thus, for antibody-drug conjugates targeting p97, antigen level and trafficking to the lysosomes are important factors for achieving robust in vitro cytotoxicity against cancer cells. Immunohistochemical analysis with L49 revealed that 62% of metastatic melanoma tumors had strong staining for p97. Overexpression of p97 in melanoma as compared with normal tissue, in conjunction with the greater sensitivity of tumor cells to L49-vcMMAF, supports further evaluation of antibody-drug conjugates for targeting p97-overexpressing tumors.

Reduction-alkylation strategies for the modification of specific monoclonal antibody disulfides.

Site-specific conjugation of small molecules and enzymes to monoclonal antibodies has broad utility in the formation of conjugates for therapeutic, diagnostic, or structural applications. Precise control over the location of conjugation would yield highly homogeneous materials that could have improved biological properties. We describe for the first time chemical reduction and oxidation methods that lead to preferential cleavage of particular monoclonal antibody interchain disulfides using the anti-CD30 IgG1 monoclonal antibody cAC10. Alkylation of the resulting cAC10 cysteine thiols with the potent antimitotic agent monomethyl auristatin E (MMAE) enabled the assignment of drug conjugation location by purification with hydrophobic interaction chromatography followed by analysis using reversed-phase HPLC and capillary electrophoresis. These analytical methods demonstrated that treating cAC10 with reducing agents such as DTT caused preferential reduction of heavy-light chain disulfides, while reoxidation of fully reduced cAC10 interchain disulfides caused preferential reformation of heavy-light chain disulfides. Following MMAE conjugation, the resulting conjugates had isomeric homogeneity as high as 60-90%, allowing for control of the distribution of molecular species. The resulting conjugates are highly active both in vitro and in vivo and are well tolerated at efficacious doses.

Generation of an intensely potent anthracycline by a monoclonal antibody-beta-galactosidase conjugate.

The L49 monoclonal antibody against the p97 antigen on melanomas and carcinomas was chemically conjugated to E. coli beta-galactosidase (beta-gal), forming a largely monomeric conjugate with preserved enzymatic activity. The resulting L49-beta-gal conjugate was used to activate (N-[(4"R,S)-4"-hexyloxy-4"-(1'''-O-beta-D-galactopyranosyl)butyl]daunorubicin) (1), a derivative of daunorubicin that has low cytotoxicity and high chemical stability. Addition of the conjugate to the prodrug resulted in an increase in cytotoxicity of approximately 10(5)-fold, a level of activation that is higher than any mAb-enzyme/prodrug combination yet described. Furthermore, the released drug had an IC(50) value of approximately 10 pM, making it significantly more potent than the vast majority of clinically approved anticancer drugs. The potential of this enzyme/prodrug combination for cancer therapy is discussed.

Design, synthesis, and in vitro evaluation of dipeptide-based antibody minor groove binder conjugates.

Antibody-drug conjugates (ADCs) were prepared consisting of DNA minor groove binder drugs (MGBs) attached to monoclonal antibodies (mAbs) through peptide linkers designed to release drugs inside the lysosomes of target cells. The site of linker attachment on the MGB was at the 5-position on the B-ring, since model studies showed that attachment of an electron-withdrawing group (i.e., acyl, carbamoyl) at this position increased the stability of the molecule. Because of the hydrophobic nature of the MGBs, several measures were required to overcome their tendencies to induce mAb aggregation upon conjugation. This is exemplified in the series of ADCs containing the amino-CBI drug 1. Initial adducts were prepared using the peptide sequence valine-citrulline, attached to a self-immolative para-aminobenzyl carbamate spacer. The resulting ADCs were completely aggregated. Removal of the self-immolative spacer, introduction of a more hydrophilic valine-lysine sequence, and incorporation of a tetraethyleneglycol unit between the mAb and the peptide resulted in conjugates that were nonaggregated, even with as many as eight drugs per mAb. These results were extended to include the hydroxy aza-CBI drug 2, which was linked to the valine-lysine sequence through a para-aminobenzyl ether self-immolative spacer. The resulting mAb conjugates were monomeric and released the hydroxy aza-CBI drug upon treatment with human cathepsin B. In vitro cytotoxicity assays established that the mAb-MGB drug conjugates were highly cytotoxic and effected immunologically specific cell kill at subsaturating doses. The results provide a general strategy for MGB prodrug design and illustrate the importance of linker hydrophilicity in making nonaggregated, active mAb-MGB conjugates.

Novel antitumor prodrugs designed for activation by matrix metalloproteinases-2 and -9.

Enzyme prodrug monotherapy takes advantage of the selectivity and specificity of enzymes that are differentially active in the immediate environment of tumor cells. Matrix metalloproteinases-2 and -9 (MMP-2 and -9, respectively) are cell-surface Zn-dependent endoproteases associated with diverse processes throughout tumor formation and progression. These enzymes have demonstrated high ratios of tumor- to nontumor-associated activity and may represent candidates for antitumor prodrug activation. Our MMP targeting strategy was to prepare and evaluate two classes of enzyme prodrugs, peptides and sequence-similar peptidomimetics, and determine which would be substrates for the enzymes and thus suitable for further in vitro and in vivo evaluation. We selected representatives of three structurally and mechanistically distinct classes of compounds for delivery, doxorubicin, several auristatins (novel synthetic members of the dolastatin class of tubulin polymerization inhibitors), and CBI-TMI (a duocarmycin class minor groove binder). The drugs were acylated on available amines with the broadly recognized MMP substrate P3-P1' sequence acetyl L-prolyl-L-leucyl-glycyl-L-leucine, or with a peptidomimetic analogue. From a panel of four peptides and four peptidomimetics, two compounds, both peptides, were found to be substrates, with specific activities in the range of 1-20 nmol min(-1) mg(-1). For MMP-9, complete conversion took place in 4-16 h; proteolysis by MMP-2 was considerably slower. Cleavage occurred, as predicted, at the Gly-Leu bond to liberate a leucyl drug, and no other intermediates or cleavage products were observed. Although the MMP-9 proteolysis products were equipotent with the parent leucyl drugs, the prodrugs were not differentially active against MMP-2 or -9-expressing versus nonexpressing cell lines during a 4 h exposure. Our data can be interpreted in light of the current understanding of the structural and mechanistic factors governing MMP-2 and -9 proteolysis.

Development of potent monoclonal antibody auristatin conjugates for cancer therapy.

We describe the in vitro and in vivo properties of monoclonal antibody (mAb)-drug conjugates consisting of the potent synthetic dolastatin 10 analogs auristatin E (AE) and monomethylauristatin E (MMAE), linked to the chimeric mAbs cBR96 (specific to Lewis Y on carcinomas) and cAC10 (specific to CD30 on hematological malignancies). The linkers used for conjugate formation included an acid-labile hydrazone and protease-sensitive dipeptides, leading to uniformly substituted conjugates that efficiently released active drug in the lysosomes of antigen-positive (Ag+) tumor cells. The peptide-linked mAb-valine-citrulline-MMAE and mAb-phenylalanine-lysine-MMAE conjugates were much more stable in buffers and plasma than the conjugates of mAb and the hydrazone of 5-benzoylvaleric acid-AE ester (AEVB). As a result, the mAb-Val-Cit-MMAE conjugates exhibited greater in vitro specificity and lower in vivo toxicity than corresponding hydrazone conjugates. In vivo studies demonstrated that the peptide-linked conjugates induced regressions and cures of established tumor xenografts with therapeutic indices as high as 60-fold. These conjugates illustrate the importance of linker technology, drug potency and conjugation methodology in developing safe and efficacious mAb-drug conjugates for cancer therapy.