Fabrice Le Gall

Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding.

Single chain variable fragments (scFv) of the murine monoclonal antibody HD37 specific to human B‐cell antigen CD19 were constructed by joining the VH and VL domains with linkers of 18, 10, 1 and 0 residues. ScFv‐18 formed monomers, dimers and small amounts of tetramers; scFv‐10 formed dimers and small amounts of tetramers; scFv‐1 formed exclusively tetramers; scFv‐0 formed exclusively trimers. The affinities of the scFv‐10 (diabody) and scFv‐1 (tetrabody) were approximately 1.5‐ and 2.5‐fold higher, respectively, than that of the scFv‐0 (triabody). The tetrabody displayed a significantly prolonged association with cell‐bound antigen (t 1/2 cell surface retention at 37°C of 26.6 min) compared to both the diabody (13.3 min) and triabody (6.7 min). This increase in avidity of the tetrabody combined with its larger size could prove to be particularly advantageous for imaging and the immunotherapy of B‐cell malignancies.

Synergistic antitumor effect of bispecific CD19 x CD3 and CD19 x CD16 diabodies in a preclinical model of non-Hodgkin's lymphoma.

To target NK cells against non-Hodgkin’s lymphoma, we constructed a bispecific diabody (BsDb) with reactivity against both human CD19 and FcγRIII (CD16). Bacterially produced CD19 × CD16 BsDb specifically interacted with both CD19+ and CD16+ cells and exhibited significantly higher apparent affinity and slower dissociation from the tumor cells than from effector cells. It was able to induce specific lysis of tumor cells in the presence of isolated human NK cells or nonfractionated PBLs. The combination of the CD19 × CD16 BsDb with a previously described CD19 × CD3 BsDb and CD28 costimulation significantly increased the lytic potential of human PBLs. Treatment of SCID mice bearing an established Burkitt’s lymphoma (5 mm in diameter) with human PBLs, CD19 × CD16 BsDb, CD19 × CD3 BsDb, and anti-CD28 mAb resulted in the complete elimination of tumors in 80% of animals. In contrast, mice receiving human PBLs in combination with either diabody alone showed only partial tumor regression. These data clearly demonstrate the synergistic effect of small recombinant bispecific molecules recruiting different populations of human effector cells to the same tumor target.

Generation and production of engineered antibodies

Various forms of recombinant monoclonal antibodies are being used increasingly, mainly for therapeutic purposes. This review specifically focuses on what is now called antibody engineering, and discusses the generation of chimeric, humanized, and fully human recombinant antibodies, immunoglobulin fragments, and artificial antigen-binding molecules. Since the production of recombinant antibodies is a limiting factor in their availability, and a shortage is expected in the future, different expression systems for recombinant antibodies and transgenic organisms as bioreactors are also discussed, along with their advantages and drawbacks.

Effect of Domain Order on the Activity of Bacterially Produced Bispecific Single-chain Fv Antibodies

Bispecific single-chain Fv antibodies comprise four covalently linked immunoglobulin variable (VH and VL) domains of two different specificities. Depending on the order of the VH and VL domains and on the length of peptides separating them, the single-chain molecule either forms two single-chain Fv (scFv) modules from the adjacent domains of the same specificity, a so-called scFv-scFv tandem [(scFv)(2)], or folds head-to-tail with the formation of a diabody-like structure, a so-called bispecific single-chain diabody (scBsDb). We generated a number of four-domain constructs composed of the same VH and VL domains specific either for human CD19 or CD3, but arranged in different orders. When expressed in bacteria, all (scFv)(2) variants appeared to be only half-functional, binding to CD19 and demonstrating no CD3-binding activity. Only the diabody-like scBsDb could bind both antigens. Comparison of the scBsDb with a structurally similar non-covalent dimer (diabody) demonstrated a stabilizing effect of the linker in the middle of the scBsDb molecule. We demonstrated that the mechanism of inactivation of CD19xCD3 diabody under physiological conditions is initiated by a dissociation of the weaker (anti-CD3) VH/VL interface followed by domain swapping with the formation of non-active homodimers. The instability of one homodimer makes the process of diabody dissociation/reassociation irreversible, thus gradually decreasing the fraction of active molecules. The structural parameters influencing the formation of functional bispecific single-chain antibodies are indicated and ways of making relatively stable bispecific molecules are proposed.

A novel tetravalent bispecific TandAb (CD30/CD16A) efficiently recruits NK cells for the lysis of CD30+ tumor cells

To improve recruitment and activation of natural killer (NK) cells to lyse tumor cells, we isolated a human anti-CD16A antibody with similar affinity for the CD16A 158F/V allotypes, but no binding to the CD16B isoform. Using CD16A-targeting Fv domains, we constructed a tetravalent bispecific CD30/CD16A tandem diabody (TandAb®) consisting solely of Fv domains. This TandAb has two binding sites for CD16A and two for CD30, the antigen identifying Hodgkin lymphoma cells. The binding and cytotoxicity of the TandAb were compared with antibodies with identical anti-CD30 domains: (1) a native IgG, (2) an IgG optimized for binding to Fc receptors, and (3) a bivalent bispecific CD30/CD16A diabody. Due to its CD16A-bivalency and reduced koff, the TandAb was retained longer on the surface of NK cells than the IgGs or the diabody. This contributed to the higher potency and efficacy of the TandAb relative to those of the other anti-CD30 antibodies. TandAb cytotoxicity was independent of the CD16A allotype, whereas the anti-CD30 IgGs were substantially less cytotoxic when NK cells with low affinity CD16A allotype were employed. TandAb activation of NK cells was strictly dependent on the presence of CD30+ target cells. Therefore, the CD30/CD16A TandAb may represent a promising therapeutic for the treatment of Hodgkin’s lymphoma; further, anti-CD16A TandAbs may function as potent immunotherapeutics that specifically recruit NK cells to destroy cancer cells.

Effect of tetravalent bispecific CD19×CD3 recombinant antibody construct and CD28 costimulation on lysis of malignant B cells from patients with chronic lymphocytic leukemia by autologous T cells

To develop an effective antitumor immunotherapy for B‐lineage non‐Hodgkins lymphoma, we constructed a tetravalent tandem diabody (tanDb) specific for both human CD19 (B‐cell marker) and CD3 (T‐cell antigen). Here, we report the effective killing of malignant primary B cells from patients with B‐cell chronic lymphocytic leukemia (B‐CLL) by autologous T cells induced by tanDb at very low E:T ratios. Mononuclear cells from patients with B‐CLL were cultured with bispecific antibody fragments in either the presence or absence of monospecific anti‐CD28 antibody. Use of tetravalent tanDbs caused almost quantitative elimination of malignant B cells from the blood samples of 19 patients and some cytotoxic activity in 3 of 23 analyzed cases. In contrast, the structurally similar but bivalent diabody and single‐chain diabody demonstrated nearly no antitumor activity in an autologous system. tanDb‐induced activation and proliferation of T cells occurred only in the presence of CD19+ target cells. Expression of the B7‐1 (CD80) and B7‐2 (CD86) molecules on the surface of leukemia cells made unnecessary the additional CD28‐costimulation of T cells. When only a few tanDb molecules were present, the effect of CD28 costimulation on T‐cell activation was more pronounced. Depending on the patient sample, we observed a 10‐ to 1,000‐fold decrease of the half‐maximal concentrations of tanDb for cell lysis. Upon CD28 crosslinking by agonistic MAb, specific tumor cell lysis was found at tanDb concentrations as low as 0.5 pM. These data demonstrate that the tetravalent CD19×CD3 tanDb might be a promising tool for the immunotherapy of human B‐cell leukemias and lymphomas.

68Ga-labelled recombinant antibody variants for immuno-PET imaging of solid tumours

PurposeRecombinant antibodies isolated from human antibody libraries have excellent affinities and high target specificity. As full-length IgGs are cleared inadequately slowly from the circulation, the aim of this work was to figure out which kind of recombinant antibody fragment proves to be appropriate for imaging epithelial cell adhesion molecule (EpCAM)-expressing tumours with the short-living radioisotope 68Ga.MethodsIn order to combine the promising tumour targeting properties of antibodies with 68Ga, four antibody variants with the same specificity and origin only differing in molecular weight were constructed for comparison. Therefore, the binding domains of a single-chain fragment variable (scFv) isolated from a human naïve antibody library were modified genetically to construct the respective full-length IgG, the tria- and diabody variants. These molecules were conjugated with the bifunctional chelating agent N,N′-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N′-diacetic acid (HBED-CC) to enable 68Ga labelling at ambient temperature and compared in biodistribution and immuno-PET imaging experiments.ResultsThe antibody variants with identical specificity proved to have the correct molecular weight, high binding affinity and specificity to their antigen, EpCAM. Radiometal complexation was efficiently performed at room temperature leading to 68Ga-labelled antibodies with unchanged binding properties compared to the original antibody variants. The best targeting properties were obtained with the scFv and especially with the diabody. The triabody showed higher absolute tumour uptake but only moderate clearance from circulation.ConclusionThe antibody variants differed considerably in normal organ uptake, clearance from circulation and tumour accumulation. The data demonstrate the feasibility of imaging solid tumours with the 68Ga-labelled diabody format. This type of recombinant protein might be a promising carrier even for the short-lived radiometal 68Ga to support e.g. the management of immunotherapy which may provide important information regarding receptor expression of solid tumours.

Abstract 3522: Preclinical development of an anti-CD30/anti-CD16A bispecific tetravalent TandAb antibody for the treatment of Hodgkin lymphoma

NK cells are professional cytotoxic immune effector cells and, unlike cytotoxic T cells, exist in a constitutively activated state not requiring pre- or co-stimulation. Thus, NK cells are attractive effectors to employ for cancer immunotherapy with bispecific antibodies. To improve the recruitment and activation of NK cells for lysis of tumor cells, we isolated a human antibody that was highly specific for the A isoform of FcαRIII (CD16A) and displayed substantially lower dissociation constant than native or Fc-optimized mAbs. The variable domains of this antibody were used to construct a tetravalent bispecific antibody, termed TandAb, consisting solely of antibody variable domains, with two valencies for CD16A (on effector cells) and two for CD30 (on target cells). CD30 represents a validated tumor target which is expressed on Hodgkin9s Reed-Sternberg lymphoma cells, and on some activated immune cells. The binding properties of the TandAb and its efficacy for inducing tumor cell lysis were compared to those of a native anti-CD30 mAb, a mAb with enhanced Fc receptor binding properties, and an anti-CD30 bispecific diabody, all of which comprised the same anti-CD30 variable domains as the TandAb. Due to its bivalency for CD16A, the TandAb was retained longer on NK cells than the IgGs or the bispecific diabody, and relatively high concentrations of IgG were necessary to displace it from NK cells. The TandAb bound equally well to both CD16A alleles (158F, 158V) indicating a potentially broader patient response than that achieved with current therapeutic mAbs. The TandAb did not induce any systemic activation of NK cells even though it9s binding was bivalent to CD16A. In a cytotoxicity assay, the TandAb displayed higher potency (EC50) and higher efficacy (maximal lysis) than the IgGs or the bispecific diabody. Cynomolgus monkeys were identified as the only relevant species for toxicity testing. Pharmacokinetics studies revealed half-lives for the TandAb up to 22.9 h after repeated dosing. No MTD was reached in a single dose escalation study to a maximum dose of 100 mg/kg. A pivotal 28 days toxicity study in which animals were dosed repeatedly up to 10 mg/kg every other day revealed an excellent safety profile. Several immunological parameters were assessed indicating no signs of immunotoxicity or systemic cytokine release. In conclusion, TandAbs may provide a more effective treatment for tumors that are refractory to treatment with IgGs and is currently being evaluated in the Phase I clinical trial. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3522. doi:1538-7445.AM2012-3522

WITHDRAWN: Tetravalent Bispecific Single-Chain Fv Antibodies for Lysis of Leukemia Cells by Autologous T Cells

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