Design and Validation of a Novel Generic Platform for the Production of Tetravalent IgG1-like Bispecific Antibodies.

We have designed and validated a novel generic platform for production of tetravalent IgG1-like chimeric bispecific Abs. The VH-CH1-hinge domains of mAb2 are fused through a peptidic linker to the N terminus of mAb1 H chain, and paired mutations at the CH1-CL interface mAb1 are introduced that force the correct pairing of the two different free L chains. Two different sets of these CH1-CL interface mutations, called CR3 and MUT4, were designed and tested, and prototypic bispecific Abs directed against CD5 and HLA-DR were produced (CD5xDR). Two different hinge sequences between mAb1 and mAb2 were also tested in the CD5xDR-CR3 or -MUT4 background, leading to bispecific Ab (BsAbs) with a more rigid or flexible structure. All four Abs produced bound with good specificity and affinity to CD5 and HLA-DR present either on the same target or on different cells. Indeed, the BsAbs were able to efficiently redirect killing of HLA-DR(+) leukemic cells by human CD5(+) cytokine-induced killer T cells. Finally, all BsAbs had a functional Fc, as shown by their capacity to activate human complement and NK cells and to mediate phagocytosis. CD5xDR-CR3 was chosen as the best format because it had overall the highest functional activity and was very stable in vitro in both neutral buffer and in serum. In vivo, CD5xDR-CR3 was shown to have significant therapeutic activity in a xenograft model of human leukemia.

Derivation and Preclinical Characterization of CYT-303, a Novel NKp46-NK Cell Engager Targeting GPC3.

Glypican-3 (GPC3) is an oncofetal antigen that is highly expressed in multiple solid tumors, including hepatocellular carcinoma, and is barely expressed in adult normal tissues except the placenta. NKp46 activation receptor is expressed in all-natural killer (NK) cells, including tumor-infiltrating NK cells. FLEX-NKTM is a platform for the production of tetravalent multifunctional antibody NK cell engagers (NKE). CYT-303 was designed using the FLEX-NK scaffold, incorporating a novel humanized NKp46 binder that does not induce NKp46 internalization and a humanized GPC3 binder that targets the membrane-proximal lobe to mediate NK cell-redirected killing of HCC tumors. CYT-303 shows sub-nanomolar binding affinities to both GPC3 and NKp46. CYT-303 was highly potent and effective in mediating NK cell-redirected cytotoxicity against multiple HCC tumor cell lines and tumor spheroids. More interestingly, it can reverse the dysfunction induced in NK cells following repeated rounds of serial killing of tumors. It also mediated antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity against GPC3-expressing HCC tumors. In vivo, CYT-303 showed no toxicity or cytokine release in cynomolgus monkeys up to the highest dose (60 mg/kg), administered weekly by intravenous infusion for 28 days. These results demonstrate the potential of CYT-303 to be a safe and effective therapy against HCC.

Antitumour effects of single or combined monoclonal antibodies directed against membrane antigens expressed by human B cells leukaemia.

BACKGROUND: The increasing availability of different monoclonal antibodies (mAbs) opens the way to more specific biologic therapy of cancer patients. However, despite the significant success of therapy in breast and ovarian carcinomas with anti-HER2 mAbs as well as in non-Hodkin B cell lymphomas with anti-CD20 mAbs, certain B cell malignancies such as B chronic lymphocytic leukaemia (B-CLL) respond poorly to anti-CD20 mAb, due to the low surface expression of this molecule. Thus, new mAbs adapted to each types of tumour will help to develop personalised mAb treatment. To this aim, we analyse the biological and therapeutic properties of three mAbs directed against the CD5, CD71 or HLA-DR molecules highly expressed on B-CLL cells. RESULTS: The three mAbs, after purification and radiolabelling demonstrated high and specific binding capacity to various human leukaemia target cells. Further in vitro analysis showed that mAb anti-CD5 induced neither growth inhibition nor apoptosis, mAb anti-CD71 induced proliferation inhibition with no early sign of cell death and mAb anti-HLA-DR induced specific cell aggregation, but without evidence of apoptosis. All three mAbs induced various degrees of ADCC by NK cells, as well as phagocytosis by macrophages. Only the anti-HLA-DR mAb induced complement mediated lysis. Coincubation of different pairs of mAbs did not significantly modify the in vitro results. In contrast with these discrete and heterogeneous in vitro effects, in vivo the three mAbs demonstrated marked anti-tumour efficacy and prolongation of mice survival in two models of SCID mice, grafted either intraperitoneally or intravenously with the CD5 transfected JOK1-5.3 cells. This cell line was derived from a human hairy cell leukaemia, a type of malignancy known to have very similar biological properties as the B-CLL, whose cells constitutively express CD5. Interestingly, the combined injection of anti-CD5 with anti-HLA-DR or with anti-CD71 led to longer mouse survival, as compared to single mAb injection, up to complete inhibition of tumour growth in 100% mice treated with both anti-HLA-DR and anti-CD5. CONCLUSIONS: Altogether these data suggest that the combined use of two mAbs, such as anti-HLA-DR and anti-CD5, may significantly enhance their therapeutic potential.

In vitro and in vivo comparison of DTPA- and DOTA-conjugated antiferritin monoclonal antibody for imaging and therapy of pancreatic cancer.

Pancreatic cancer has a very poor prognosis with a less than 5% survival rate at 5 years. Neither external beam radiation nor chemotherapy, alone or in combination, have given encouraging results so far. A possible solution might come from the use of targeted therapy such as radioimmunotherapy. We present here the results obtained from the preclinical development of a new monoclonal antiferritin antibody (Ab), AMB8LK. Ferritin is overexpressed in pancreatic cancer and could thus be used as a target for the delivery of radioactivity at the tumour sites. The AMB8LK Ab was conjugated to three chelating agents: the 2-(4-isothiocyanatobenzyl)-diethylenetriamine pentaacetic acid (PSCN-Bz-DTPA), the (R)-2-amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid (p5CN-Bz-CHX-A"-DTPA) and the 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (pSCN-Bz-DOTA). Radiolabelling of the three immunoconjugates with indium 111 and yttrium 90 as well as in vitro stability and immunoreactivity against pure ferritin and cells expressing ferritin were analysed. In vivo biodistribution studies were conducted on normal and on human pancreatic adenocarcinoma CAPAN-1 tumour bearing mice. These experiments demonstrated good radiolabelling (>95%), stability and immunoreactivity of the three compounds. In the biodistribution studies, differences between the three immunoconjugates were apparent in the rate of blood clearance and in tumour, liver and bone uptake. A very good pancreatic adenocarcinoma tumour targeting was observed especially with the Bz-DTPA-AMB8LK: 20% of the injected dose of the indium-labelled compound 3 days after injection; 15% of the injected dose 5 days after that of the yttrium-labelled Ab. Altogether, these results in animal models suggest that (90)Y-Bz-DTPA-AMB8LK is a good candidate for further therapeutic efficacy studies.

The design and evaluation of a novel targeted drug delivery system using cationic emulsion-antibody conjugates.

In an attempt to design a targeted drug delivery system to tumors' over-expressing H-ferritin specifically recognized by a monoclonal antibody, AMB8LK, a cationic emulsion - AMB8LK conjugate was prepared. A novel cross-linker molecule bearing maleimide group was synthesized and added to cationic emulsion formulation for AMB8LK Fab' fragment covalent coupling. NMR spectroscopy confirmed the cross-linker synthesis and the preservation of the active maleimide function. SDS gel-electrophoresis results corroborated the formation of the Fab' fragment. Different densities of Fab' fragments (10-200 Fab'/oil droplet) were conjugated to emulsion droplet interface and no changes in the physico-chemical properties were observed ( approximately 120 nm size and zeta potential of approximately +30 mV). The coupling efficiency ranged from 55% to 70% and was visualized by TEM showing gold particles attached to the droplet interface. Cell culture studies demonstrated specific binding to cells as confirmed by the occurrence of the marked reduction in binding when free AMB8LK Mab was incubated before adding the AMB8LK-emulsion conjugate to the cells. The coupling of AMB8LK Fab' fragment to the cationic emulsion increased the cells uptake by 50% as compared to non-conjugated respective cationic emulsion. Appropriate conditions were, thus, identified for coupling AMB8LK Fab' fragment to cationic emulsion without altering the specificity and affinity of the Mab fragment to the tumor antigen.

A novel antiangiogenic and vascular normalization therapy targeted against human CD160 receptor.

Angiogenesis plays an essential role in several diseases of the eye and in the growth of solid tumors, but existing antiangiogenic therapies have limited benefits in several cases. We report the antiangiogenic effects of a monoclonal antibody, CL1-R2, in several animal models of neovascularization. CL1-R2 recognizes human CD160, a membrane receptor which is conserved in various mammal species. We show that CD160 is expressed on the endothelial cells of newly formed blood vessels in human colon carcinoma and mouse B16 melanoma but not in vessels of healthy tissues. CL1-R2 reduced fibroblast growth factor 2-induced neovascularization in the rabbit cornea, in a mouse model of oxygen-induced retinopathy, and in a mouse Matrigel plug assay. Treatment of B16 melanoma-bearing mice with CL1-R2 combined with cyclophosphamide chemotherapy caused regression of the tumor vasculature and normalization of the remaining vessels as shown by Doppler ultrasonography, intravital microscopy, and histology. These studies validate CD160 as a potential new target in cases of human pathological ocular and tumor neoangiogenesis that do not respond or become resistant to existing antiangiogenic drugs.

A quantitative evaluation of the molecular binding affinity between a monoclonal antibody conjugated to a nanoparticle and an antigen by surface plasmon resonance.

We have designed a site-specific drug colloidal carrier ultimately for improving pancreatic and lung cancer treatment. It is based on a nanoparticulate drug delivery system that targets tumors overexpressing H-ferritin. A monoclonal antibody, AMB8LK, specifically recognizing H-ferritin was thiolated and conjugated to maleimide-activated polylactide nanoparticles (NPs) resulting in the formation of immunonanoparticles (immunoNPs). The AMB8LK immunoNPs exhibited a mean diameter size of 112+/-20nm and a density of 76 antibody molecules per NP. AMB8LK immunoNPs were evaluated for uptake and binding properties on CAPAN-1 and A-549 cell lines, using confocal microscopy. ImmunoNPs demonstrated specific binding and increased uptake of the desired cells by means of monoclonal antibodies (MAbs), compared to nonconjugated NPs. A lipophilic paclitaxel derivative, paclitaxel palmitate (pcpl), was encapsulated within the various NP formulations, and their cytotoxic effect was evaluated on A-549 cells using MTT assay. Pcpl-loaded AMB8LK immunoNPs showed a significantly increased cytotoxic effect when compared to pcpl solution and pcpl NPs. Surface plasmon resonance (SPR) was used to determine quantitatively the affinity constants of native AMB8LK and AMB8LK immunoNPs to gain insight on the affinity of the MAbs following the conjugation process onto NPs. The results of the association/dissociation and affinity kinetics of the interaction between H-ferritin and native AMB8LK or AMB8LK immunoNPs revealed similar constant values, showing that the conjugation process of the MAb to the NPs did not alter the intrinsic specificity and affinity of the MAb to the antigen. In conclusion, at the cellular level, AMB8LK immunoNPs may carry drugs to desired overexpressing antigen cells with adequate affinity properties, potentially leading to improved drug therapy and reduced systemic adverse effects.

Overcoming the formulation obstacles towards targeted chemotherapy: in vitro and in vivo evaluation of cytotoxic drug loaded immunonanoparticles.

The aim of this study was to design a new one step conjugation of monoclonal antibodies (MAbs) to surface activated pegylated polyester nanoparticles (NPs) and evaluate the pharmacokinetic profile and therapeutic effect of paclitaxel palmitate (pcpl) loaded anti-HER2 immunoNPs in mice as compared to pcpl solution and NPs following IV injection. The density of the antibody conjugated to the NPs was found to be around 35 MAbs/NP (70% coupling efficiency). In vitro cell culture studies showed good binding and uptake results when immunoNPs were incubated with PC-3 and CAPAN-1 cell lines. Both pcpl NPs and immunoNPs showed significant increased t1/2, C(max) and AUC values as compared to the values of pcpl solution in mice. There was no significant difference in the C(max) and AUC values between pcpl NPs and pcpl immunoNPs. However, the immunoNPs concentrated much less in the liver and spleen than NPs. The pharmacokinetic behavior of the immunoNPs was markedly different from the pharmacokinetic profile of the naked MAb showing that the MAb lost its intrinsic molecular pharmacokinetic properties following conjugation to the NPs. The immunoNPs elicited a significant anti-tumor activity as compared to the pcpl solution and NPs, although the tumor growth was not fully inhibited.

Radioimmunotherapy of refractory or relapsed Hodgkin's lymphoma with 90Y-labelled antiferritin antibody.

The aim of this study was to evaluate the safety and efficacy of radiolabelled rabbit polyclonal antiferritin antibody in relapsed or refractory Hodgkin's lymphoma. The protocol included a first intravenous injection of In-labelled antiferritin antibody, followed by immunoscintigraphy at 4, 48 and 72 h, and an intravenous injection of Y-labelled antiferritin antibody in the case of tumour targeting. Ten patients were included in the study: median number of chemotherapy regimens: 3; number of autografted patients: 8; number of previously irradiated patients: 9; response to last chemotherapy: six partial response and four progressions. All immunoscintigraphies showed tumour targeting. Nine patients were treated, as the last patient died from progressive Hodgkin's lymphoma before therapeutic injection. Median injected activity was 12 MBq/kg (0.32 mCi/kg). Among the 10 patients who were included in the study, one complete response and six partial responses were observed (overall response rate 70%) with a median duration of response of 8 months (range: 7-12 months). Toxicity was mainly haematological, with grade 1 or 2 neutropenia and anaemia, and grade 2 and 3 thrombocytopenia. The pharmacokinetic study showed that the half-lives of In and Y were almost identical. These results confirm those previously reported and show the therapeutic potential of rabbit polyclonal antiferritin antibody in relapsed or refractory Hodgkin's lymphoma. They therefore justify further multicentre prospective trials.