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Radioimmunotherapy (RIT) is a method for selectively delivering radionuclides to cancer cells while reducing the radiation dose to normal tissues. However, because of slow clearance of MAbs, normal tissues also received radiotoxicity. One of the promising strategies is linking on-demand cleavable (ODC) peptides between radiometal chelates and the tumor targeting agents. We have tested this proof-of-concept by using ODC peptides that are designed to be cleaved only by TNKase and are resistant to cleavage by enzymes present in the plasma and the tumor. TNKase-specific peptide linkers using l- and d-amino acids were screened by OBOC combinatorial peptide libraries. One of the best peptides was linked to radiometal chelate and ChL6-MAb to prepare radioimmunoconjugate (RIC). Optimization and characterization of the linker conjugation to MAb show (a) 1-2 peptides linked to each MAb; (b) immunoreactivity >80%; (c) specific activity of the RIC 0.7-1 microCi/microg; (d) RIC stable over 7 days in human plasma; and (e) radiometal-chelated ODC peptide cleaved from the RIC in plasma by TNKase at clinical dose levels of 10 microg/ml. The percent release of radiochelate from RIC was 50% at 24h and 85% over 7 2h in vitro. This novel ODC-linked RIC could be a potential molecule for RIT.

Dextran and PEG-coated iron oxide nanoparticles (NP), when suitably modified to enable conjugation with molecular targeting agents, provide opportunities to target cancer cells. Monoclonal antibodies, scFv, and peptides conjugated to 20 nm NP have been reported to target cancer for imaging and alternating magnetic field (AMF) therapy. The physical characteristics of NPs can affect their in vivo performance. Surface morphology, surface charge density, and particle size are considered important factors that determine pharmacokinetics, toxicity, and biodistribution. New NanoFerrite (NF) particles having improved specific AMF absorption rates and diameters of 30 and 100 nm were studied to evaluate the variation in their in vitro and in vivo characteristics in comparison to the previously studied 20 nm superparamagnetic iron oxide (SPIO) NP. SPIO NP 20 nm and NF NP 30 and 100 nm were conjugated to (111)In-DOTA-ChL6, a radioimmunoconjugate. Radioimmunoconjugates were conjugated to NPs using 25 microg of RIC/mg of NP by carbodiimide chemistry. The radioimmunonanoparticles (RINP) were purified and characterized by PAGE, cellulose acetate electrophoresis (CAE), live cell binding assays, and pharmacokinetics in athymic mice bearing human breast cancer (HBT 3477) xenografts. RINP (2.2 mg) were injected iv and whole body; blood and tissue data were collected at 4, 24, and 48 h. The preparations used for animal study were90% monomeric by PAGE and CAE. The immunoreactivity of the RINP was 40-60% compared to (111)In-ChL6. Specific activities of the doses were 20-25 microCi/2.2 mg and 6-11 microg of mAb/2.2 mg of NP. Mean tumor uptakes (% ID/g +/- SD) of each SPIO 20 nm, NF 30 nm, and 100 nm RINP at 48 h were 9.00 +/- 0.8 (20 nm), 3.0 +/- 0.3 (30 nm), and 4.5 +/- 0.8 (100 nm), respectively; the ranges of tissue uptakes were liver (16-32 +/- 1-8), kidney (7.0-15 +/- 1), spleen (8-17 +/- 3-8), lymph nodes 5-6 +/- 1-2), and lung (2.0-4 +/- 0.1-2). In conclusion, this study demonstrated that 100 nm NF NP could be conjugated to (111)In-mAb so that the resulting RINP had characteristics suitable for AMF therapy. Although 100 nm RINP targeted tumor less than 20 nm SPIO RINP, their heating capacity is typically 6 times greater, suggesting the 100 nm NF RINP could still deliver better therapy with AMF.

Immunotherapies for cancer offer attractive alternatives to conventional therapies although human anti-globulin antibody (HAGA) against the antibody (Ab) administered to the patient can be an obstacle to repeated treatment. Monoclonal antibodies (mAb), whether foreign or human in origin, have been used safely in patients for two decades. Adverse events have not proven to be significant clinical obstacles, although alterations of pharmacokinetic behavior of subsequently administered Ab can lead to less effective therapy. Not only is HAGA safe, but it can be associated with beneficial immunity in patients. Studies have shown that some patients have unexpectedly prolonged survival associated with HAGA. In our own non-Hodgkin's lymphoma (NHL) patients treated with mouse Lym-1 anti-lymphoma mAb, a high human anti-mouse Ab (HAMA) titer was associated with increased survival. The possible mechanisms linking HAMA responses to survival are likely related to Ab generated against the idiotopes of the administered Ab. An induced immune cascade in these patients, including anti-idiotypic Ab (Ab2) and cytotoxic Ab1' or Ab3 probably contributed to survival. In summary, HAGA should not a priori preclude the therapeutic use of Ab for cancer.

BACKGROUND Radioimmunotherapy (RIT) is a promising new modality for targeted, systemic delivery of radionuclides specifically to sites of androgen-independent metastatic prostate cancer. To be effective, RIT requires an antibody with specificity for malignant cells and appropriate pharmacokinetics in the body. METHODS Specific binding of the L6 monoclonal antibody to prostate cancer cell lines or cell lysates was determined by enzyme-linked immunoabsorbent assay (ELISA), solid-phase radioimmunoassay, and immunofluorescent staining. Biodistribution, tumor uptake, and whole body and blood clearances of 125I-L6 were determined in nude mice bearing human prostate cancer xenografts. RESULTS The L6 monoclonal antibody showed strong binding to the lysates of PC3 and DU145 prostate cancer cell lines, and 66% binding to live PC3 cells. The L6 antibody specifically targeted prostate cancer in PC3 and DU145-tumored nude mice, where approximately 10% of the injected dose of 125I-L6 bound to prostate cancer. Low-normal organ uptake was found, and the blood clearances were similar in each group of tumored mice. CONCLUSIONS The L6 monoclonal antibody targets human prostate cancer xenografts in nude mice and has low-normal organ uptake. Therefore, further study of the radiolabeled L6 monoclonal antibody for RIT of prostate cancer is warranted. Prostate 37:91–97, 1998. © 1998 Wiley-Liss, Inc.

A patient with aggressive, chemotherapy-resistant non-Hodgkins lymphoma (NHL) was treated with 131I-Lym-1, a mouse antibody, on a protocol designed for serial therapy. Human anti-mouse antibody (HAMA) developed within 1 month of initial therapy. The patient also developed an antibody to the hypervariable region of the Lym-1 antibody (Lym-1 specific). Because the patient was responding to therapy, plasmaphoresis was used to reduce the level of HAMA followed by unlabeled Lym-1 calculated to be sufficient to block residual HAMA. This allowed additional therapy on three subsequent occasions over 5 months. Despite very high HAMA levels, no untoward effects from administrations of Lym-1 were observed. The HAMA response of the patient included anti-Lym-1 specific antibodies containing anti-idiotypic antibodies. The anti-Lym-1 antibody level has been sustained over the 9 year interval since 131I-Lym-1 therapy and has been associated with a uniquely long remission of the patient's disease. These observations demonstrate that, under certain circumstances, radioimmunotherapy (RIT) can be given safely and effectively despite HAMA. Anti-idiotypic antibodies could have induced an immune cascade that contributed to the prolonged disease-free survival of the patient.

Host development of human anti-mouse antibodies (HAMA) in response to administered antibodies has been reported as a problem for antibody imaging and therapy. However, radioimmunotherapy has been shown to be effective in patients with B-cell malignancies because their immunodeficient state precludes or delays development of a HAMA response to mouse antibodies. Baseline HAMA activity was assayed in 60 patients with B-lymphocytic non-Hodgkin's lymphoma or chronic lymphocytic leukemia and sequentially in 43 patients who were subsequently treated with radiolabeled Lym-1 antibody. Pre-existing “HAMA” activity was found in 3 (5%) of the 60 patients screened for treatment consideration. The incidence of development of HAMA in the 43 patients treated with multiple doses of radiolabeled Lym-1 antibody was 12 (28%). There was no evidence for an anaphylactoid or related response in the HAMA positive patients. HAMA activity interrupted therapy in 14% of the patients (6 of 43) but did not preclude therapeutic responses to radiolabeled Lym-1 therapy. Median survival for the HAMA positive patients was longer (18 months) than for those who did not develop HAMA activity (9 months).

Summary Fifty-eight per cent of patients with B-cell malignancies had durable responses to treatment with 131I-Lym-1. Myelosuppression manifested by peripheral blood cytopenia was the radiation dose-limiting toxicity. The mean biologic half-times were 3.3 and 31.2 h for the fast and slow phases, respectively, of the blood clearance and 33.5 h for the clearance from the total body. Nonpenetrating radiation from the blood contributed 0.18 rad and penetrating radiations from the total body contributed 0.18 rad per administered mCi to the bone marrow. The average total contribution from both of these sources was 0.36 ± 0.14 rad mCi-1. Clearances and marrow radiation doses were remarkably constant among different patients and among different therapy doses for the same patient. These results are potentially useful as an initial approximation for other mouse monoclonal antibodies of the same isotype. While radiation to normal marrow from 'spill-over' incident to specific targeting of 131I-Lym-1 on malignant B-cells in the marrow is not addressed in this publication because it is unique for each patient, it should be considered in the case of individual patients.

To mimic the molecular specificity and cell selectivity of monoclonal antibody (mAb) binding while decreasing size, nanomolecules (selective high-affinity ligands; SHALs), based on in silico modeling, have been created to bind to human leukocyte antigen-DR (HLA-DR10), a signaling receptor protein upregulated on the malignant B-lymphocytes of non-Hodgkin's lymphoma and chronic lymphocytic leukemia. SHALs were synthesized with a biotin or DOTA chelate (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid), using a solid-phase lysine-polyethyleneglycol backbone to link sets of ligands shown previously to bind to HLA-DR10. Using cell-binding and death assays and confocal microscopy, SHAL uptake, residualization, and cytocidal activity were evaluated in HLA-DR10 expressing and nonexpressing live, human lymphoma cell lines. All of the SHALs tested were selective for, and accumulated in, expressing cells. Reflecting binding to HLA-DR10 inside the cells, SHALs having the Ct ligand (3-(2-([3-chloro-5-trifluoromethyl)-2-pyridinyl]oxy)-anilino)-3-oxopropanionic acid) residualized in expressing cells greater than 179 times more than accountable by cell-surface membrane HLA-DR10. Confocal microscopy confirmed the intracellular residualization of these SHALs. Importantly, SHALs with a Ct ligand had direct cytocidal activity, similar in potency to that of Lym-1 mAb and rituximab, selectively for HLA-DR10 expressing lymphoma cells and xenografts. The results show that SHALs containing the Ct ligand residualize intracellularly and have cytocidal effects mediated by HLA-DR10. These SHALs have extraordinary potential as novel molecules for the selective targeting of lymphoma and leukemia for molecular therapy and imaging. Further, these SHALs can be used to transport and residualize cytotoxic agents near critical sites inside these malignant cells.

Background A variety of arginine-rich peptide sequences similar to those found in viral proteins have been conjugated to other molecules to facilitate their transport into the cytoplasm and nucleus of targeted cells. The selective high affinity ligand (SHAL) (DvLPBaPPP)2LLDo, which was developed to bind only to cells expressing HLA-DR10, has been conjugated to one of these peptide transduction domains, hexa-arginine, to assess the impact of the peptide on SHAL uptake and internalization by Raji cells, a B-cell lymphoma. Results An analog of the SHAL (DvLPBaPPP)2LLDo containing a hexa-arginine peptide was created by adding six D-arginine residues sequentially to a lysine inserted in the SHAL's linker. SHAL binding, internalization and residualization by Raji cells expressing HLA-DR10 were examined using whole cell binding assays and confocal microscopy. Raji cells were observed to bind two fold more 111In-labeled hexa-arginine SHAL analog than Raji cells treated with the parent SHAL. Three fold more hexa-arginine SHAL remained associated with the Raji cells after washing, suggesting that the peptide also enhanced residualization of the 111In transported into cells. Confocal microscopy showed both SHALs localized in the cytoplasm of Raji cells, whereas a fraction of the hexa-arginine SHAL localized in the nucleus. Conclusion The incorporation of a hexa-D-arginine peptide into the linker of the SHAL (DvLPBaPPP)2LLDo enhanced both the uptake and residualization of the SHAL analog by Raji cells. In contrast to the abundant cell surface binding observed with Lym-1 antibody, the majority of (DvLPBaPPP)2LArg6AcLLDo and the parent SHAL were internalized. Some of the internalized hexa-arginine SHAL analog was also associated with the nucleus. These results demonstrate that several important SHAL properties, including uptake, internalization, retention and possibly intracellular distribution, can be enhanced or modified by conjugating the SHALs to a short polypeptide.

Nanoparticle thermotherapy (NPTT) uses monoclonal antibody-linked iron oxide magnetic nanoparticles (bioprobes) for the tumor-specific thermotherapy of cancer by hysteretic heating of the magnetic component of the probes through an externally applied alternating magnetic field (AMF). The present study investigated the effect of NPTT on a human prostate cancer cell line, DU145. The concept of total heat dose (THD) as a measure for NPTT was validated on a cellular level and THD was correlated to cell death in vitro. The study, furthermore, explored the potential enhancement of the NPTT effect through added external beam radiation therapy (EBRT), because both forms of treatment have a different, and potentially complementary, mechanism of causing cell death.Using carbodiimide, (111)In-DOTA-ChL6 was conjugated to dextran iron oxide 20-nm particles with polyethylene glycol COOH groups on the surface and purified as (111)In-bioprobes. NPTT and EBRT were applied alone and combined to cells labeled with the bioprobes. Cell response was monitored by measuring lactate dehydrogenase (LDH), a product of cytolysis, in the medium. This distinct focus on the response to NPTT was possible, since we found in previous studies that the LDH assay was relatively insensitive to the response of cells (without bioprobes) to EBRT in the dose levels given here.NPTT showed a significantly increased cell death at a total calculated heat dose of 14.51 and 29.02 J/g cells (50% and 100% AMF duty, 350 Oe, 136 kHz, 12 cycles, 20 minutes total), compared with AMF exposure in the absence of bioprobes. Adding EBRT to NPTT did not increase cell death, as measured by LDH. However, EBRT given to cells labeled with bioprobes caused significant cell death at radiation doses of 10 Gy and higher.In human prostate cancer cell cultures, NPTT applied as a single modality caused cell death that correlated with THD estimation; complete cell death occurred at 14.51 J/g cells. Consequently, enhancement of the NPTT effect through the addition of EBRT could not be addressed. Interestingly, EBRT induced cell death on bioprobe-labeled cells at EBRT levels that did not show cell death in the absence of bioprobes; this phenomenon is worth investigating further.

Purpose: More than two decades of research and clinical trials have shown radioimmunotherapy to be a promising approach for treating various forms of cancer. Lym-1 antibody, which binds selectively to HLA-DR10 on malignant B-cell lymphocytes, has proved to be effective in delivering radionuclides to non–Hodgkin's lymphoma and leukemia. Using a new approach to create small synthetic molecules that mimic the targeting properties of the Lym-1 antibody, a prototype, selective high-affinity ligand (SHAL), has been developed to bind to a unique region located within the Lym-1 epitope on HLA-DR10. Experimental Design: Computer docking methods were used to predict two sets of small molecules that bind to neighboring cavities on the β subunit of HLA-DR10 surrounding a critical amino acid in the epitope, and the ligands were confirmed to bind to the protein by nuclear magnetic resonance spectroscopy. Pairs of these molecules were then chemically linked together to produce a series of bidentate and bisbidentate SHALs. Results: These SHALs bind with nanomolar to picomolar Kd's only to cell lines expressing HLA-DR10. Analyses of biopsy sections obtained from patients also confirmed that SHAL bound to both small and large cell non–Hodgkin's lymphomas mimicking the selectivity of Lym-1. Conclusions: These results show that synthetic molecules less than 1/50th the mass of an antibody can be designed to exhibit strong binding to subtle structural features on cell surface proteins similar to those recognized by antibodies. This approach offers great potential for developing small molecule therapeutics that target other types of cancer and disease.

Despite their large size, antibodies have proven to be suitable radioisotope carriers to deliver systemic radiotherapy, often molecular image-based, for lymphoma and leukemia. To mimic antibody (Ab) targeting behavior while decreasing size by 50-100x, a combination of computational and experimental methods were used to generate molecules that bind to unique sites within the HLA-DR epitopic region of Lym-1, an Ab shown effective in patients. Lym-1 Ab mimics (synthetic high afinity ligands; SHALs) were generated and studied in vitro, using live cell binding assays, and/or pharmacokinetic studies over 24 h in xenografted mice given 1 or 20 microg SHAL doses i.v. Multimilligram amounts of each of the dimeric (bis) SHALs were synthesized at high purity, and labeled with indium-111 at high specific activity and purity. These SHALs were selective for HLA-DR and HLA-DR expressing malignant cells and had functional affinities that ranged from 10(-9) M (nanomolar) to 10(-10) M. Blood clearances ranged from 3.6 to 9.5 h and body clearances ranged from 15.2 to 43.0 h for the 6 bis DOTA-SHALs studied in a mouse model for non-Hodgkin's lymphoma (NHL). While localization was shown in Raji NHL xenografts, biodistribution was influenced by 'sinks' for individual ligands of the SHALs. Highly pure, dimeric mimics for HLA-DR Ab were synthesized, biotinylated and radiolabeled, and showed selectivity in vitro. Pharmacokinetic behavior in mice was influenced by the ligands and by the linker length of the dimeric SHALs. Nanomolar or better functional affinity was observed when a suitably long linker was used to connect the two bidentate SHALs. The concept and methodology are of interest because applicable for targeting most proteins; the SHAL synthetic platform is highly efficient and adaptive.

Despite their large size, antibodies (Abs) are suitable carriers to deliver systemic radiotherapy, often molecular image-based, for lymphoma and leukemia. Lym-1 Ab has proven to be an effective radioisotope carrier, even in small amounts, for targeting human leukocyte antigen DR (HLA-DR), a surface membrane protein overexpressed on B-cell lymphoma. Pairs of molecules (referred to as ligands), shown by computational and experimental methods to bind to each of 2 sites within the Lym-1 epitopic region, have been linked to generate small (kDa) molecules (referred to as selective high-affinity ligands [SHALs]) to mimic the targeting properties of Lym-1 Ab. Methods: A lysine-polyethylene glycol (PEG) backbone was used to synthetically link 2 of the following ligands: deoxycholate, 5-leuenkephalin, triiodothyronine, thyronine, dabsyl-L-valine, and N-benzoyl-L-arginyl-4amino-benzoic acid to generate a series of 13 bidentate SHALs with a biotin or 1,4,7,10-tetraazacyclododecane-NN',N '',N'''-tetraacetic acid (DOTA) chelate attached to the linker. These SHALs have been assessed for their selectivity in binding to HLA-DR10-expressing cells and for their pharmacokinetics and tissue biodistribution in mice. Biotinylated versions of these SHALs discriminated cell lines positive for HLA-DR10 expression with near-nanomolar affinity. The DOTA versions of 4 SHALs were labeled with In-111 for pharmacokinetic studies in mice with HLA-DR10-expressing malignant Raji xenografts. Results: The bidentate, biotinylated, and DOTA-SHALs were synthesized in high-purity, multimilligram amounts. Mean radiochemical and product yields and purities were 90%, 75%, and 90% at mean specific activities of 3.9 MBq/mu g (105 mu Ci/mu g) for the In-111-labeled SHALs. As expected, rapid blood clearance and tumor targeting were observed. The pharmacokinetics of the SHALs was influenced by the component ligands. Biliary clearance, kidney localization, and serum receptor binding contributed to less favorable tumor targeting. Conclusion: A series of SHALs was readily synthesized in multimilligram amounts and showed the expected selective binding in vitro. Better selection of the SHAL components should provide second-generation SHALs with improved properties to fulfill the substantial potential of these novel molecular carriers for targeting.

Background: About one-third of patients with relapsed B-cell malignancies develop human anti-mouse antibody (HAMA) following mouse antibody treatment. The purpose of this study was to assess the relationship between HAMA and survival in patients given a mouse anti-lymphoma monoclonal antibody (mAb), Lym-1, directed against a unique epitope of HLA-DR antigen that is up-regulated on malignant B-cells. Methods: ELISA was used to quantify HAMA in 51 patients with B-cell malignancies treated with iodine-131 (131I) labeled Lym-1. Sera were collected prior to and following radioimmunotherapy (RIT) with 131I-Lym-1 until documented to be HAMA negative or throughout lifetime. Univariate, then multivariate analyses including other risk factors, were used to analyze the relationship of HAMA to survival. The relationships of HAMA to prior chemotherapies and to absolute lymphocyte counts prior to RIT were also assessed. Results: Eighteen of 51 patients (35%) developed HAMA following RIT (range of ultimate maximum titers, 6.6–1,802 μg/ml). Using the time dependent Cox proportional hazards model, maximum HAMA titers were associated with survival (P=0.02). HAMA continued to be significant for survival in multivariate analyses that included known risk factors. In Landmark analysis of 39 patients that survived at least 16 weeks, median survival of patients with HAMA less than 5 μg/ml was 61 versus 103 weeks for patients with HAMA equal or greater than 5 μg/ml at 16 weeks (P=0.02). The median survival of the five patients with highest maximum HAMA titers was 244 weeks. At 16 weeks, there was an inverse correlation between the maximum HAMA titer and the number of previous chemotherapies (P

The purpose of this study was to examine idiotypic cascade mechanisms in the plasma of a prolonged survivor patient with aggressive non-Hodgkin's lymphoma (NHL). It is a follow-up to previously published seminal studies by this laboratory showing survival benefit associated with radioimmunotherapy in NHL patients. Immunoglobulin from the patient's plasma was purified, characterized, and shown to possess the activities expected of idiotypic antibodies.Plasma from a NHL patient treated with Lym-1 was precipitated with ammonium sulfate and octanoic acid, followed by immunoadsorbant chromatography with solid phase Lym-1 monoclonal antibody to purify Ab2. The last purification step involved the binding of Ab3 to glutaraldehyde-fixed Raji cells, followed by acid elution of Ab3. Proteins were quantified and characterized. Antibody-dependent cellular cytotoxicity activity was determined using a standard (51)Cr release assay.Purified immunoglobulin populations exhibited the characteristics of Ab2beta and Ab3 antibodies. Both showed ability to compete with the binding of Lym-1 to its tumor cell target, and Ab3 showed ability to induce antibody-dependent cellular cytotoxicity.This study offers direct evidence for initiation of a multilevel idiotypic cascade in a patient undergoing passive monoclonal antibody therapy for NHL. The patient's prolonged disease-free survival may, thus, be understood in the context of the generation of endogenous, self-perpetuating tumor-specific antibodies.

The selection and characterization of anti-MUC-1 single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer molecules designed for optimal blood clearance and tumor penetration. The mucin MUC-1 was chosen as an antigen because it is abundantly expressed on epithelial cancers in an aberrantly glycosylated form, making it structurally and antigenically distinct from MUC-1 expressed on normal cells.A previously constructed anti-MUC-1 phage display library from hyperimmunized mice, with 5 x 10(5) calculated variants, was screened for the selection of anti-MUC-1 scFvs. Selection criteria were high binding to a MUC-1 peptide containing 4 tandem repeats of 20 amino acids and to MUC-1-positive MCF-7 (human breast cancer) cell lysates in ELISA.Six anti-MUC-1 scFv clones were selected and characterized. Nucleotide sequencing showed that four of them were full length scFv genes (variable heavy chain + variable light chain), whereas the remaining two contained either a variable heavy chain or a variable light chain alone. Their binding affinities (K(a)) range between 8 x 10(7) and 10(9) M(-1). Immunohistopathology demonstrated reactivity with breast cancer cells (MCF-7 and BT20) and human breast biopsy tissue. Molecular modeling revealed high structural similarity of the anti-MUC-1 scFvs with the X-ray-determined structure of the anti-CEA scFv (MFE-23).In vitro antigen binding was demonstrated for the selected anti-MUC-1 scFvs. The binding affinities of these scFvs are in a promising range for efficient in vivo antigen binding. These anti-MUC-1 scFvs will be evaluated as antigen-binding modules in new multifunctional agents for the detection and therapy of cancer.

Rapid blood and body clearances have hampered effective tumor targeting by small molecules. We used branched poly(ethylene glycol) (pegylated) polymers (M(r) 40,000, M(r) 70,000, M(r) 100,000, and M(r) 150,000) conjugated to tumor-specific and control peptides to assess the effect of both molecular weight and tumor specificity on pharmacokinetics and biodistribution.Pegylated specific lymphoma-binding peptide and control peptide (containing stereoisomers of proline and aspartate) were synthesized, radiolabeled with (111)In, fractionated by size, and injected into Raji lymphoma-bearing athymic mice (4-6 mice/group). Pharmacokinetics were followed for 2 days to evaluate effects of specificity and molecular size on blood clearance, body clearance, and biodistribution.As molecular size increased, blood and body clearances decreased (P0.001). The effect of molecular size on blood clearance was not altered by ligand binding specificity (P = 0.21), with t(1/2) ranging from 5.4 h (M(r) 40,000) to 17.7 h (M(r) 150,000). However, ligand specificity did alter body clearance, with pegylated control peptides clearing the body more slowly than pegylated specific peptides [P = 0.03; range, 19.1-91.3 h (specific peptides) versus 23.6-115.7 h (control peptides)]. At 24 h, there was more uptake of specific versus control pegylated peptides in tumor, liver, and marrow, but there was less uptake in kidneys, with a more pronounced difference for the higher molecular weight peptides (P0.01).These results demonstrate that the pharmacokinetics and biodistribution of peptides and resultant uptake in tumor and normal tissues can be altered by both molecular size and ligand specificity, with molecular size affecting pharmacokinetics and organ uptake in a predictable manner.

Immunotherapeutic approaches to cancer offer an attractive adjunct to conventional modalities, although human antiglobulin responses can be an obstacle to repeated treatment. This study of a large number of patients with B-cell malignancies, over an extended period of time, characterized their human antimouse antibody (HAMA) seroconversion.A total of 617 samples from 112 subjects were analyzed for HAMA titers. Eighty-five patients with B-cell malignancies; 12 breast cancer patients, and 15 volunteers were titered for comparison. Fifty-six B-cell malignancy patients were titered for HAMA throughout Lym-1 radioimmunotherapy (RIT); 29 were titered after a single imaging dose of Lym-1 antibody.Baseline titers did not correlate with subsequent HAMA seroconversion against Lym-1. Only 1 of 29 (3%) of the patients developed HAMA after an imaging dose of Lym-1. Of the RIT trial group, 37 of 56 (66%) never developed HAMA above baseline despite multiple doses. Of those who did (19 of 56; or 34%), the HAMA responses fell into two categories. Thirteen responded rapidly (median of 31 days) and were termed "early responders," whereas 6, termed "late responders," had a median response time of 111 days. Early responders developed higher peak HAMA titers with fewer exposures to Lym-1 and took longer to return to baseline than did the late responders. The frequency of new antiglobulin seroconversion decreased as the number of exposures increased.Seventy-seven percent of B-cell malignancy patients developed no response or a weak response after multiple doses of mouse Lym-1 antibody. Positive responders occurred in all histology types and fell into two categories differing in seroconversion time and titer, possibly indicative of the initial state of the immune system.

SH7139, our most advanced small molecule therapeutic for non-Hodgkin's lymphoma, was designed to bind to a unique site on HLA-DR10, a cell surface receptor found on B-cell lymphocytes and over-expressed on B-cell derived malignancies. Studies conducted with lymphoma and other cell lines have shown that SH7139 binds selectively and is highly cytotoxic only to tumor cells expressing HLA-DR10. A previous study also showed SH7139 to be highly effective in treating human Burkitt's lymphoma (Raji) in a mouse xenograft model. We have used this same mouse model to compare the efficacy of SH7139 delivered by different routes and using different treatment regimens. The results show that the greatest survival times were achieved with mice given SH7139 by i.p. injection on days 0, 7 and 14. Doses delivered i.p. on days 0, 2 and 4 and orally on days 0,7,14,21,28 and 35 were slightly less effective, and a single dose delivered i.p. on day 0 and three doses delivered orally on days 0, 7, and 14 were the least effective. The concentration of SH7139 that achieved these effects in vivo, which was determined by measuring xenograft uptake of 111-In-labeled SH7139, was found to be in the sub-nanomolar range. In addition to identifying the best route and dosing regimen for future studies and confirming the drug works in vivo at concentrations similar to those found to be effective in cells cultured in vitro, these studies also show the drug can be effective when administered orally. Citation Format: Rodney Balhorn, Gary Mirick, Gerald L. DeNardo, Laurel Beckett, Judy Li, Saphon Hok, Monique Balhorn. Effect of route and dosing regimen on efficacy of SH7139 in mouse Burkitt's lymphoma xenografts. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2703. doi:10.1158/1538-7445.AM2014-2703

Selective high affinity ligands (SHALs) are novel targeting agents composed of lysine/mini-polyethylene glycol scaffolds linking two or three ‘recognition’ ligands together. The tridentate SHAL SH7139 is selectively cytotoxic at sub-nanomolar concentrations to lymphoma cells over-expressing HLA-DR10. Electron microscopy examinations of Burkitt's lymphoma xenografts from mice treated with SH7139 suggest one mechanism for cell killing may involve cell signaling-induced apoptosis analogous to that observed with Lym-1 antibody. An alternative mechanism is that intracellular hydrolysis of the SHAL's amide bonds following its uptake and metabolism by lymphoma cells could lead to the release of the three recognition ligands and the subsequent inhibition (or promotion) of cellular activities that trigger apoptosis or induce cell death and necrosis. Each of the three recognition ligands (Dv, Ct, and Cb) is independently, but non-selectively, toxic to both Raji (HLA-DR10 positive) and Jurkat (HLA-DR10 negative) cells. The Ct ligand, 3-(2-([3-chloro-5-trifluoromethyl)-2-pyridinyl]oxy)-anilino)-3-oxopropanionic acid, is a structural analog of aryloxyphenoxypropionate herbicides that inhibit acetyl-CoA carboxylase (ACC), a key enzyme in fatty acid biosynthesis that is upregulated in many cancers to meet their need for rapid growth and proliferation. Our studies show that although Ct could be accommodated into the herbicide binding pocket of ACC, this ligand was not found to inhibit human and rat ACC1 or ACC2. However, metabolic cleavage of the single amide bond present in Ct would lead to the production of two smaller metabolites, an aniline derivative and malonate, both of which are known to be biologically active and potentially toxic compounds. The Dv ligand, dabsyl-L-valine, belongs to a family of biphenylsulfonamides, many of which have been shown to inhibit matrix metalloproteinases such as MMP9 and MMP14 that are upregulated in tumor cells. The Cb ligand, 4-[4-(4-chlorobenzyl)piperazino]-3-nitrobenzene carboxylic acid, is a structural analog of a large number of inhibitors that have been shown to block the assembly of the contractile ring and the initiation of cytokinesis by inhibiting guanosine nucleoside binding to MgcRacGAP, a protein that participates in the activation of the myosin motor and the initiation of cleavage furrow ingression. A combination of SH7139 metabolism studies, computer modeling, and enzyme inhibition assays have been performed to determine if Ct, Dv and Cb and their metabolites are produced and whether they contribute to lymphoma cell killing. Citation Format: Monique C. Balhorn, Gary Mirick, Dong Cheng, Zhengping Ma, Edmond Y. Lau, Saphon Hok, Gerald L. DeNardo, Rod Balhorn. Intracellular uptake and metabolism of SH7139: Is it a targeted prodrug for B-cell lymphomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5460. doi:10.1158/1538-7445.AM2014-5460

The Lym-1 monoclonal antibody was conjugated with the bifunctional chelating agent 6-[p-(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane-N,N ',N'',N'''-tetraacetic acid (BAT), using 2IT as a linker, and radiolabeled with 67Cu to make the radiopharmaceutical, 67Cu-2IT-BAT-Lym-1. Ten patients received a total of 18 doses of 67Cu-2IT-BAT-Lym-1 as targeted, systemic radiotherapy. The beta phase of blood clearance, when corrected for 67Cu decay, was positive or flat, a phenomenon not observed in similar patients treated with 131I-Lym-1. The flat beta phase of blood clearance suggested recycling of 67Cu from 67Cu-2IT-BAT-Lym-1 to another plasma protein. Therefore, the amount of 67Cu transferred from the radiopharmaceutical to CP, Alb, and TF was measured using affinity-purified polyclonal antibodies. The fraction of plasma 67Cu precipitated by anti-human CP increased daily; most blood radioactivity was 67Cu-CP after a median of 4 days (range 2-7 days). The transfer of 67Cu to CP was observed in all patients and was consistent from dose to dose within the same patient. An average of 2.8 +/- 1.5% (range 0.8-7.8%) of the 67Cu dose (%ID) was transferred to CP. The release rate of 67Cu-CP from the liver into the blood was 0.9 +/- 0.4 %ID/day for the first 3 days. The 67Cu-CP effective clearance half-life was 3.7 +/- 0.7 days. Subtraction of the 67Cu-CP activity from the total blood radioactivity yielded a biphasic blood clearance similar to that obtained for patients given 131I-Lym-1. Cu-67-CP increased the AUC for whole blood by 24 +/- 10%. The %ID of 67Cu recycled correlated with GGT, ALT, and alkaline phosphatase levels; r = 0.958 (p0.001), 0.857 (p0.01), and 0.822 (p0.01), respectively. Albumin levels correlated negatively with recycled copper (r = -0.745, p0.05). The data suggest that the liver metabolizes 67Cu-2IT-BAT-Lym-1 and recycles a small fraction of the 67Cu, transferring it to CP.

Lym-1, a monoclonal antibody (MAb) that preferentially targets malignant lymphocytes, has induced therapeutic remissions in patients with advanced non-Hodgkin's lymphoma (NHL) or chronic lymphocytic leukemia (CLL) when labeled with iodine-131 (131I). Based on the strategy of fractionating the total radiation dose, trials were designed to define the safety, toxicity, and efficacy of a series of doses of 131I-Lym-1 given 2-6 weeks apart. All patients had disease resistant to standard therapy. 131I-Lym-1 was given after unconjugated Lym-1 and the 131I dose was escalated in Phase I-II trials. Therapy proved safe. The dose-limiting toxicity was thrombocytopenia. Nonhematological toxicities did not exceed grade 2 except for infrequent instances of grade 3 hypotension. In a low-dose (LD) trial of 131I-Lym-1, tumor regression occurred in 25 (83%) of 30 patients and 17 (57 %) had durable remissions; 3 of the remissions were complete. In a maximum tolerated dose (MTD) trial of 131I-Lym-1, 10 (71%) of 14 entries that received at least two doses of 131I-Lym-1 therapy and 11 (52%) of 21 total entries had remissions; 7 of the remissions were complete. All 3 entries in the MTD cohort of 100 mCi/m2 [3.7 MBq/m2] of body surface area had durable complete remissions. Therapeutic remission and human anti-mouse antibody (HAMA) after Lym-1 therapy were associated with increased survival that was significant in multivariate analyses. Evidence for an Ab3 idiotypic network with an antibody cytotoxic for Raji human lymphoma was found in the only patient examined in detail thus far; this patient was studied because she had a high titer, HAMA and prolonged survival. In conclusion, 131I-Lym-1 induced durable remissions in patients with chemotherapy-resistant NHL or CLL and was associated with acceptable toxicity. In a subset of the patients, survival was quite prolonged perhaps related to development of Ab3.

Like rituximab, monoclonal antibodies reactive with human leukocyte antigen have potent antilymphoma activity. However, size limits their vascular and tissue pene- tration. To mimic monoclonal antibody binding, nanomolecules have been synthesized, shown specific for the s subunit of HLA-DR10, and selective for cells expressing this protein. Selective high affinity ligands (SHALs) containing the 3-(2- ((3-chloro-5-trifluoromethyl)-2-pyridinyl)oxy)-anilino)-3- oxopropanionic acid (Ct) ligand residualized and had anti- lymphoma activity against expressing cells. Herein, we show the extraordinary potency in mice with human lymphoma xenografts of a tridentate SHAL containing this ligand. After titrating antilymphoma activity in cell culture, a randomized preclinical study of a tridentate SHAL containing the Ct ligand was conducted in mice with established and aggressive human lymphoma xenografts. Mice having HLA-DR10 expressing Raji B- or Jurkat's T-lymphoma xenografts were randomly assigned to receive either treatment with SHAL at a dose of 100 ng i.p. weekly for 3 consecutive weeks, or to be untreated. Primary end-points were cure, overall response rates and survival. Toxicity was also evaluated in these mice, and a USFDA general safety study was conducted in healthy Balb/c mice. In Raji cell culture, the threshold and IC50 concentrations for cytotoxic activity were 0.7 and 2.5 nmol (pm/ml media), respectively. When compared to treated Jurkat's xenografts or untreated xenografts, Raji xenografts treated with the SHAL showed an 85% reduction in hazard of death (P=0.014; 95% confidence interval 32-95% reduction). There was no evidence for toxicity even after i.p. doses 2000 times greater than the treatment dose associated with cure of a majority of the mice with Raji xenografts. When compared with control groups, treatment selectively improved response rates and survival in mice with HLA-DR10 expressing human lymphoma xenografts at doses not associated with adverse events and readily achievable in patients.