Safety assessment and dose selection for first-in-human clinical trials with immunomodulatory monoclonal antibodies.

Modulating immune responses with monoclonal antibodies (mAbs) that target immune molecules has become a promising therapeutic strategy and is under investigation for the treatment of cancer and (auto)-immune diseases. A major hurdle to the development and early clinical investigation of many immunomodulatory mAbs is the inherent risk of adverse immune-mediated drug reactions in humans, such as cytokine storms, autoimmunity, and immunosuppression. Dose selection for first-in-human (FIH) clinical trials involving immunomodulatory mAbs, and mAbs in general, is based on specifically designed preclinical safety studies, primarily in nonhuman primates (NHPs), and on mechanistic ex vivo investigations. Dose selection in such trials is challenging for a number of reasons related to safety. In this context, safety-relevant differences between NHP and human immune systems, species selection/qualification and preclinical study design considerations, the receptor occupancy model and its calculation, the minimal anticipated biological effect level (MABEL) and its use in the selection of a safe starting dose in humans, microdosing and the impact of immunogenicity on safety assessment of mAbs, and safety-relevant formulation properties of therapeutic mAbs are critically reviewed. In addition, the current regulatory requirements are presented and discussed to demonstrate how the TeGenero TGN1412 case is leading to increased regulatory scrutiny regarding dose selection for FIH clinical trials.

TACI attenuates antibody production costimulated by BAFF-R and CD40.

B cell activating factor of the TNF family (BAFF), plays critical roles in B cell survival, activation, differentiation, and antibody (Ab) production. BAFF binds to three receptors: BAFF-R, transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) and B cell maturation antigen. While BAFF-R is the primary receptor for B cell costimulation by BAFF, TACI is reported to serve as a positive or negative regulator for B cell responses depending on conditions. To determine the real role of TACI in B cell responses, we examined the functional relationship between TACI and BAFF-R in Ab production from human peripheral blood B cells using agonistic mAb. BAFF-R and CD40 enhanced IgG secretion and B cell proliferation, which were inhibited by TACI. Although TACI induced mild B cell apoptosis, its extent did not correlate with that of TACI-mediated inhibition of IgG secretion. In addition, TACI inhibited B-lymphocyte-induced maturation protein-1 expression, IgG secretion from previously IgG-negative selected B cells, and activation-induced cytidine deaminase expression enhanced by BAFF-R and CD40. Importantly, BAFF-R and CD40 enhanced B cell responsiveness to TACI-mediated suppression. Thus, BAFF may attenuate T cell-independent and -dependent B cell responses by TACI.

A monoclonal V kappa l light chain responsible for incomplete proximal tubulopathy.

Calcium and phosphate metabolism abnormalities are frequent in myeloma patients and the role of renal lesions in such ionic perturbations may have been overlooked. The authors herein report the complete primary structure of a Bence Jones Vkappal light chain responsible for myeloma-associated proximal tubulopathy with increased phosphaturia. Plasma and serum biochemical evaluations indicated a proximal tubular dysfunction mainly manifested as tubular acidosis and phosphate loss. The study of a kidney biopsy showed interstitial and tubular lesions with numerous myeloma casts and peculiar features of the proximal tubular cells, which carried numerous phagolysosomal inclusions with occasional crystalline periodic striation. The nephrotoxic light chain primary structure was deduced from the bone marrow monoclonal plasma cells RNA. The kappal sequence was highly homologous to kappa chains previously characterized in patients with Fanconi syndrome. It was related to the Vkappal subgroup and was composed of a variable segment encoded by the O8/O18 germline gene rearranged to Jkappa4. The primary sequence presented unusual features restricted to the variable region, including substitutions of residues 28 and 31 in the complementary determining region 1 (CDR1) by amino acids of different charge. An unusual conformation of the kappal domain, likely resulting from somatic hypermutation, could alter the catabolism of the protein after its internalization and result in the tubular cell dysfunction. Comparison with Fanconi syndrome studies suggests that Vkappal Bence Jones proteins may damage proximal tubular cells to an extent varying according to light chain (LC) sequence and structure, either leading to crystal formation and Fanconi syndrome or inducing partial inhibition of proximal tubule function.

Influence of cytokines, monoclonal antibodies and chemotherapeutic drugs on epithelial cell adhesion molecule (EpCAM) and LewisY antigen expression.

MoAbs against tumour-associated antigens (TAA) may be useful for the treatment of colorectal cancer. Since an increased expression of TAA may lead to enhanced antibody-dependent cellular cytotoxicity we examined whether the cytokines IL-2, IL-4, IL-6, IL-10, IL-12, interferon-alpha (IFN-alpha), IFN-gamma, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor and tumour necrosis factor-alpha can influence EpCAM and LewisY expression on the surface of the colorectal carcinoma cell lines HT29, LoVo and SW480. We found that only IFN-alpha increased significantly whereas IL-4 decreased both EpCAM and LewisY expression. IFN-gamma significantly increased LewisY expression only. When tumour cells were treated with MoAb, the LewisY-specific MoAb BR55-2 down-regulated LewisY antigen expression, whereas MoAb 17-1A, which binds to EpCAM, up-regulated this TAA after 3 days of culture. The cytokines IFN-alpha or IFN-gamma combined with MoAb 17-1A enhanced further slightly the expression of EpCAM. In additional experiments with chemotherapeutic drugs commonly used for the treatment of colorectal cancer, we found that 5-fluorouracil, mitomycin-C and oxaliplatin up-regulated EpCAM and LewisY antigen expression. Raltitrexed enhanced LewisY and down-regulated EpCAM expression, whereas CPT-11 had no influence at all. The highest expression for EpCAM on HT29 cells was achieved by the combination of IFN-alpha, 5-fluorouracil and MoAb 17-1A. Our results may be useful for defining combinations of biological and chemotherapeutic drugs for the treatment of colorectal cancer. Further trials should evaluate to what extent these combinations enhance antibody-dependent cellular cytotoxicity.

A novel glycosylphosphatidyl inositol-anchored protein on human leukocytes: a possible role for regulation of neutrophil adherence and migration.

We report here a novel glycosylphosphatidyl-inositol (GPI)-anchored glycoprotein on human leukocytes. Treatment of neutrophils with a mAb (3H9) to this molecule sequentially up-regulates and down-regulates beta2 integrin-dependent adhesion of these cells as well as their transendothelial migration in vitro. In addition, this mAb simultaneously modulates the avidity of beta2 integrin for its ligand, iC3b, with kinetics similar to those observed in 3H9 modulation of neutrophil adherence. This mAb also induces beta2 integrin-dependent cytoskeletal remodeling. This novel GPI-anchored protein (GPI-80) is highly homologous with Vanin-1, a recently reported GPI-anchored protein that is expressed on perivascular thymic stromal cells and is involved in thymus homing in mice. The finding that both GPI-80 and Vanin-1 are 40% homologous with human biotinidase suggests the existence of a biotinidase superfamily of molecules that may be involved in the regulation of leukocyte trafficking.

Patterns of costimulation of T cell clones by cross-linking CD3, CD4/CD8, and class I MHC molecules.

The ability of mAb to class I MHC molecules, CD3, or CD4/CD8 to stimulate human T cell clones alone or in combination was examined. Cross-linking each of these surface Ag with appropriate mAb and goat anti-mouse Ig (GaMIg) resulted in a unique pattern of increase in intracellular free calcium ([Ca2+]i) and different degrees of functional activation. Cross-linking class I MHC molecules provided the most effective stimulus of IL-2 production and proliferation. Cross-linking more than one surface Ag induced a compound calcium signal with characteristics of each individual response. Cross-linking CD3 + HLA-A,B,C caused a rapid and prolonged increase in [Ca2+]i and synergistically increased IL-2 production and proliferation of all clones. Cross-linking CD3 + CD4/CD8 also generated a compound calcium signal and increased IL-2 production and DNA synthesis. Purposeful inclusion of CD3 was not required for costimulation as cross-linking HLA-A,B,C + CD4/CD8 also increased [Ca2+]i, IL-2 production, and proliferation. Cross-linking three surface Ag, CD3 + HLA-A,B,C + CD4/CD8, resulted in the greatest initial and sustained [Ca2+]i, IL-2 production, and DNA synthesis. Although there was a tendency for the various stimuli to increase both [Ca2+]i and functional responsiveness, neither the magnitude nor duration of the increased [Ca2+]i correlated with the amount of IL-2 produced or the ultimate proliferative response. To determine whether costimulation required that the various surface molecules were cross-linked together, experiments were carried out using isotype specific secondary antibodies. Augmentation of [Ca2+]i and costimulation of functional responses were noted when class I MHC molecules were cross-linked and CD3 was bound, but not cross-linked. Similarly, costimulation through CD3 and CD4/CD8 was observed when CD4/CD8 was cross-linked and the CD3 complex was engaged by an anti-CD3 mAb which was not further cross-linked. In contrast, costimulation by class I MHC molecules and CD4/CD8 was only observed when these molecules were cross-linked together. These data demonstrate that cross-linking class I MHC determinants or CD4/CD8 provides a direct signal to T cell clones that can be enhanced when CD3 is independently engaged. The results also indicate that T cell clones can be stimulated without engaging CD3 by the combination of signals delivered via class I MHC molecules and CD4/CD8, but only when these determinants were cross-linked together. These studies have demonstrated that these cell surface molecules differ in their capacity to deliver activation signals to T cell clones and also exhibit unique patterns of positive cooperativity in signaling potential.

Enhancement of human B-cell proliferation by a monoclonal antibody to CD43.

Monoclonal antibodies (MoAb) to human leucocyte sialoglycoprotein, CD43, have been shown to deliver mitogenic signals to human T cells or to enhance T-cell proliferation induced by concanavalin A, anti-CD3 antibodies or phorbol ester. In this paper, we studied the effects of anti-CD43 MoAb B1B6 on the activation of human B cells. Anti-CD43 MoAb B1B6 was not mitogenic by itself for human B cells. However, when added together with TPA, both resting and in vivo activated tonsillar B cells, containing 5-10% and about 35% CD43+ respectively, responded with three- to fivefold higher proliferation compared to that obtained with TPA alone. A peak in the proliferative response was reached on day 3. Optimal proliferation was obtained when the antibody was present from the start of culturing. Addition of MoAb B1B6 together with a calcium ionophore, ionomycin, did not induce B-cell proliferation. Neither did mAb B1B6 sustain the growth of B cells that were already in the cell cycle, i.e. precultured with phorbol ester (PDB) and ionomycin for 3 days. The results are similar to those obtained with antibodies to CD22 and CD23 and show that early progression signals are delivered to resting B cells through CD43 in the presence of primary activators of protein kinase C.

Identification and localization of allergenic determinants on grass group I antigens using monoclonal antibodies.

Epitopes recognized by five mAb which block the binding of human IgE antibodies to grass group I (GpI) Ag were characterized and partially mapped. Site specificity studies defined four apparently non-overlapping blocking antibody binding sites on the meadow fescue GpI molecule, Fes e I. One of these sites (site A) was localized to a 14,000 m.w. fragment designated P3 generated by CNBr cleavage of purified Fes e I. The P3 peptide possessed human IgE binding sites as well as other epitopes (non-site A) defined by 19 other anti-GpI mAb. All of the P3 reactive antibodies recognized cross-reactive determinants found on GpI Ag isolated from five different grasses suggesting that P3 is a conserved portion of grass GpI molecules. The P3 fragment from Fes e I was used to immunize mice and induced antibodies which reacted with intact GpI Ag from all 5 different grasses currently being studied in this laboratory.

Monoclonal antibodies against leucoagglutinin-reactive human T-lymphocyte surface components. II. Studies on the mechanism of K46M-induced activation and determination of the frequency of responding cells.

A monoclonal antibody, K46M (IgM kappa), obtained after immunization with leucoagglutinin (La)-reactive T-cell surface components, stimulated human lymphocytes to proliferate. It induced maximal proliferation at greater than 20 micrograms IgM/ml after 3-4 days of culture. Cells stimulated by K46M produced interleukin 2 (IL-2) and gamma interferon (IFN-gamma) and expressed receptors for IL-2 and transferrin. The majority of the activated cells were phenotypically T cells as defined by monoclonal antibodies against CD3 and CD2, and an increase in the K46M-positive cells was also observed during the activation period. K46M-activated cells display major histocompatibility complex (MHC)-unrestricted cytotoxicity against several cultured target cells. The frequencies of the cytotoxic and of the proliferative precursor cells were determined using a limiting dilution assay. K46M seems to activate a larger fraction of cytotoxic precursor cells against Molt 4 than against K562, but the statistical significance of these observations requires further exploration. Both K46M or La activated 40% of PBL to proliferate, whereas 70% of PBL were induced by OKT3. However, the frequency of K46M-activated cells was 40% only when the lymphocytes were plated at low cell densities, i.e. less than 0.5 cells per well. At higher densities an inhibition of proliferation was seen that resulted in a biphasic response curve, indicating that the activation of PBL by K46M was not a single hit event. This was not found with either La or OKT3. Whether K46M, in contrast to OKT3 and La, activates a subpopulation with suppressor activity remains to be established.

B cell stimulatory factor 1 induces lobster agglutinin 1-separated mouse thymocytes to express cytotoxic T lymphocyte activity.

Mouse thymocytes low in surface sialic acid were prepared by using the lectin lobster agglutinin 1 (LAg1). These LAg1-thymocytes do not become CTL when incubated with Con A or Con A plus mouse rIL-2, whereas unseparated thymocytes and thymocytes with high levels of surface sialic acid develop good levels of polyclonal CTL activity under these conditions. However, LAg1- thymocytes developed high levels of CTL activity when incubated with B cell stimulatory factor-1 (BSF-1), provided as the supernatant of the rBSF-1-secreting T cell hybridoma D9-C1.12.17. Affinity-purified BSF-1 from D9-C1 supernatant and rBSF-1 also stimulated these cells to become CTL, but they were not as active as the D9-C1 supernatant. The ability of D9-C1 supernatant and of affinity-purified BSF-1 to induce CTL activity was inhibited by the anti-BSF-1 mAb 11B11. Moreover, this mAb inhibited the ability of 24-h Con A-stimulated spleen cell supernatant to induce these cells to express CTL activity. 11B11 also inhibited LAg1+ thymocytes from becoming CTL when stimulated with Con A alone. These experiments suggest that BSF-1 is required for LAg1- and LAg1+ thymocytes to become CTL.

A regulatory role for the CD4 and CD8 molecules in T cell activation.

The role of the CD4 and CD8 molecules in T cell activation is presently a matter of controversy. Although their role as associative binding elements to MHC class II or class I is well documented, their influence on the triggering process in unclear. Because antibodies to CD4 or CD8 block T cell activation in the absence of their respective ligands, a negative signaling by these molecules has been suggested. However, recent experimental evidence argues against a negative regulatory effect of these molecules, since, e.g., simultaneous cross-linking of TCR and CD4 leads to enhanced T cell activation. Therefore, a current model suggests that the association of TCR and CD4 in the membrane gives a positive signal essential for triggering. In this report we present evidence that this model is likely to be too simple. Anti-CD4 and CD8 antibodies inhibit alternative, nonreceptor pathways of T cell triggering via Tp103 and Tp44 in the absence of class II positive accessory or target cells. These antibodies also inhibit bypass activation of T cells by phorbol ester and calcium ionophore in an accessory cell-free system. Furthermore, if the CD4 or CD8 molecules are removed from the cell surface by antibody-induced modulation, the proliferative and cytotoxic response of T cell clones is enhanced. This enhancement is also observed if resting peripheral blood T cells are used as responder cells. These data show that the CD4 or CD8 molecules have a complex regulatory function in T cell activation beyond the requirement for co-cross-linking with the TCR.

Monoclonal antibodies block the bromelain-mediated release of human placental alkaline phosphatase from cultured cancer cells.

Certain monoclonal antibodies (mAbs) to human placental alkaline phosphatase (PLAP) block bromelain cleavage of a 2-kDa segment from each of the two polypeptide chains of PLAP. These mAbs also prevent the release of PLAP from cultured cancer cell surfaces by bromelain. Such proteolysis-blocking mAbs serve as tools to specifically modify the molecular topography of cell surfaces by protease treatment.

Thy-1 antigen expression by murine high-proliferative capacity hematopoietic progenitor cells. I. Relation between sensitivity to depletion by Thy-1 antibody and stem cell generation potential.

Hematopoietic stem cells of high proliferative potential such as the giant macrophage colony-forming cell HPP-CFC, were present in the marrow of mice treated with high dose 5-fluorouracil (5Fu) (150 mg/kg i.v.), whereas most committed granulocyte-macrophage progenitors, GM-CFU-C, were depleted. Enrichment of primitive stem cells in post 5-Fu bone marrow (5FuBM) was reflected in an enhanced capacity to proliferate in suspension cultures stimulated by the mixture of lymphokines present in Con A spleen-conditioned medium supernatant (Con A CM) when compared to normal bone marrow. The population of blast-like cells harvested at 5 days from suspension cultures of 5FuBM with Con A CM showed marked increases in stem cells GM-CFU-C and HPP-CFC. For this reason, 5FuBM was utilized to study the cell surface characteristics of putative pluripotential stem cells capable of giving rise to committed stem cells in suspension cultures. Treatment of 5FuBM (BDF1 mice) before suspension culture with a high concentration of either of two cytotoxic monoclonal antibodies directed against the Thy-1.2 surface antigen in the presence of rabbit complement reduced or abrogated the generation of stem cells HPP-CFC and GM-CFU-C in suspension cultures, even though the input content of HPP-CFC and GM-CFU-C in treated 5FuBM compared with control 5FuBM showed little reduction by the antibody plus complement treatment. The Thy-1+ cell required for generation of stem cells was not a T cell, because reconstitution of Thy-1.2-depleted 5FuBM with spleen nylon nonadherent (T) cells did not reconstitute the generation of stem cells, even though T cells did grow in the suspension cultures. In addition, depletion from 5FuBM of cells expressing Lyt-1 and Lyt-2 antigens, unambiguous markers of T cell-thymocyte differentiation, did not ablate the generation of HPP-CFC and GM-CFU-C. Rather, performance of Thy-1 cell depletion at lower efficiency, which still abrogated T cell function, ablated generation of HPP-CFC but did not affect the generation of GM-CFU-C. It was concluded that 5FuBM contains distinct Thy-1+ primitive stem cells expressing different amounts of Thy-1 antigen correlating with their respective generation potentials. Some of these Thy-1+ progenitor cells may be pluripotential.