In Vitro and In Vivo Evaluation of Cysteine and Site Specific Conjugated Herceptin Antibody-Drug Conjugates

Antibody drug conjugates (ADCs) are monoclonal antibodies designed to deliver a cytotoxic drug selectively to antigen expressing cells. Several components of an ADC including the selection of the antibody, the linker, the cytotoxic drug payload and the site of attachment used to attach the drug to the antibody are critical to the activity and development of the ADC.The cytotoxic drugs or payloads used to make ADCs are typically conjugated to the antibody through cysteine or lysine residues. This results in ADCs that have a heterogeneous number of drugs per antibody. The number of drugs per antibody commonly referred to as the drug to antibody ratio (DAR), can vary between 0 and 8 drugs for a IgG₁ antibody. Antibodies with 0 drugs are ineffective and compete with the ADC for binding to the antigen expressing cells. Antibodies with 8 drugs per antibody have reduced in vivo stability, which may contribute to non target related toxicities.In these studies we incorporated a non-natural amino acid, para acetyl phenylalanine, at two unique sites within an antibody against Her2/neu. We covalently attached a cytotoxic drug to these sites to form an ADC which contains two drugs per antibody.We report the results from the first direct preclinical comparison of a site specific non-natural amino acid anti-Her2 ADC and a cysteine conjugated anti-Her2 ADC. We report that the site specific non-natural amino acid anti-Her2 ADCs have superior in vitro serum stability and preclinical toxicology profile in rats as compared to the cysteine conjugated anti-Her2 ADCs. We also demonstrate that the site specific non-natural amino acid anti-Her2 ADCs maintain their in vitro potency and in vivo efficacy against Her2 expressing human tumor cell lines. Our data suggests that site specific non-natural amino acid ADCs may have a superior therapeutic window than cysteine conjugated ADCs.     

The effect of antigen concentration,antibody valency and size,and tumor architecture on antibody binding in multicell spheroids

Intact IgG₁ and F(ab′)₂ anti-carcinoembryonic antigen antibodies penetrate human colon adenocarcinoma multicell spheroids much more slowly than Fab fragments and the molecular weight and the binding site valency appear to be the most important factor in determining the rate of penetration. The rate is also influenced considerably by the number of antigen binding sites per cell, with a high antigen concentration slowing penetration appreciably. The tumor cell architecture appears to have a minor effect on antibody penetration when compared to antibody size or antigen concentration.     

Kinetic analysis of biphasic protein modification reactions cooperative effects

A mathematical treatment of protein modification reactions is presented, and it is shown thai in these cases protein modification is described by a summation of exponential functions of reaction time, the number of exponentials being equal to the number of modified protein species. It is shown that in cases of protein modification cooperativity, there is a strict dependence of the coefficients of the multiexponential modification equation on the constants of the same equation. The conditions necessary for a reduction of a multiexponential protein modification equation to one of a summation of two exponentials only are examined. The possible formulae for the coefficients of a two-exponential-summation equation, used to describe the modification of protein models with two, three or four modifiable residues (as well as some aspects of models with five and six modifiable residues) per protein molecule are derived. It is seen that the number of such coefficients is severely limited. The most frequently obtained formula for the lower stoichiomelric coefficient of a 'wo-exponential-summation equation is Ak_a/(k_a-k_b). where k_b and k_b are the constants of the two exponentials of the equation, and A is a constant. The value most frequently arrived at for A is (n?1)/n, where n is the number of modifiable residues per protein molecule, while values such as 1/n, or a/n (where a is an integer, and also where a < n) are also possible. In most of the cooperative protein modification models worked out, k_a is identical with k_n, viz., k_a is identical with the rate constant for the first stoichiometric protein modification.     

Allowance for antibody bivalence in the determination of association rate constants by kinetic exclusion assay

This investigation completes the amendment of theoretical expressions for the characterization of antigen–antibody interactions by kinetic exclusion assay—an endeavor that has been marred by inadequate allowance for the consequences of antibody bivalence in its uptake by the affinity matrix (immobilized antigen) that is used to ascertain the fraction of free antibody sites in a solution with defined total concentrations of antigen and antibody. A simple illustration of reacted site probability considerations in action confirms that the square root of the fluorescence response ratio, R_Ag/R_o, needs to be taken in order to determine the fraction of unoccupied antibody sites, which is the parameter employed to describe the kinetics of antigen uptake in the mixture of antigen and antibody with defined initial composition. The approximately 2-fold underestimation of the association rate constant (k_a) that emanates from the usual practice of omitting the square root factor gives rise to a corresponding overestimate of the equilibrium dissociation constant (K_d)—a situation that is also encountered in the thermodynamic characterization of antigen–antibody interactions by kinetic exclusion assay.     

Theoretical studies of clonal selection: minimal antibody repertoire size and reliability of self-non-self discrimination.

Viewing the immune system as a molecular recognition device designed to identify “foreign shapes”, we estimate the probability that an immune system with N_Ab monospecific antibodies in its repertoire can recognize a random foreign antigen. Furthermore, we estimate the improvement in recognition if antibodies are multispecific rather than monospecific. From our probabilistic model we conclude: (1) clonal selection is feasible, i.e. with a finite number of antibodies an animal can recognize an effectively infinite number of antigens; (2) there should not be great differences in the specificities of antibody molecules among different species; (3) the region of a foreign molecule recognized by an antibody must be severely limited in extent; (4) the probability of recognizing a foreign molecule, P, increases with the antibody repertoire size N_Ab; however, below a certain value of N_Ab the immune system would be very ineffectual, while beyond some high value of N_Ab further increases in N_Ab yield diminishing small increases in P; (5) multispecificity is equivalent to a modest increase (probably less than 10) in the antibody repertoire size N_Ab, but this increase can substantially improve the probability of an immune system recognizing a foreign molecule.Besides recognizing foreign molecules, the immune system must distinguish them from self molecules. Using the mathematical theory of reliability we argue that multisite recognition is a more reliable method of distinguishing between molecules than single site recognition. This may have been an important evolutionary consideration in the selection of weak non-covalent interactions as the basis of antigen-antibody bonds.     

An immunostimulatory substance in the ascites fluid of patients with cancer metastatic to peritoneum.

The ascitic fluids from patients with cancer metastatic to the peritoneum contain a factor(s) which stimulates the primary antibody response to sheep red blood cells (SRBC) in vitro. This enhancement is manifested by an increase in the number of plaque-forming cells per culture and a slight increase in plaque size. This factor has a molecular weight in the range 30,000–100,000 as determined by Sephadex gel filtration. The factor, which we have called “stimulatory factor” (SF), will completely replace the requirement for fetal calf serum in the Mishell-Dutton type of assay. Enhancement of the antibody response is most apparent at suboptimal culture conditions. SF does not increase the number of plaque-forming cells to the T-independent antigen Escherichia coli but there is a marked increase in the size of the plaques produced to the lipopolysaccharide using coated SRBC as targets. The stimulation induced by this factor is not due to endotoxin contamination since endotoxin is heat stable and the SF is heat inactivated at 80 °C for 10 min. In addition endotoxin does not act in a manner similar to SF. Thus, the SF appears to influence both T and B cells. With thymus-dependent antigen the factor results in increased numbers of antibody cells being generated; with thymus-independent antigen the factor results in increased quantity of antibody being produced.     

Limiting Factors in Photosynthesis: II. IRON STRESS DIMINISHES PHOTOCHEMICAL CAPACITY BY REDUCING THE NUMBER OF PHOTOSYNTHETIC UNITS

It has been proposed that Fe stress may be used in the study of limiting factors in photosynthesis as an experimental means of varying photochemical capacity in vivo (Plant Physiol 1980 65: 114-120). In this paper the effect of Fe stress on photosynthetic unit number, size, and composition was investigated by measuring P₇₀₀, cytochrome (Cyt) f, chlorophyll (Chl) a, and Chl b in sugar beet leaves. The results show that when Fe stress reduced Chl per unit area by 80% (from 60 to 12 micrograms per square centimeter), it decreased the number of P₇₀₀ molecules per unit area by 88% and Cyt f per unit area by 86%; over the same range the Chl to P₇₀₀ ratio increased by 37% but there was no significant change in the Chl to Cyt f ratio. These data suggest that Fe stress decreases photochemical capacity and Chl per unit area by diminishing the number of photosynthetic units per unit leaf area.     

Methylglyoxal-modified arginine residues — a signal for receptor-mediated endocytosis and degradation of proteins by monocytic THP-1 cells

Non-enzymatic glycosylation or glycation of proteins to form advanced glycation endproducts (AGE) has been proposed as a process which provides a signal for the degradation of proteins. Despite this, the AGE which act a recognition factor for receptor-mediated endocytosis and degradation of glycated proteins by monocytes and macrophages has not been identified. Methylglyoxal, a reactive α-oxoaldehyde and physiological metabolite, reacted irreversibly with arginine residues in proteins to form N_δ-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine and N_δ-(5-methyl-4-imidazolon-2-yl)ornithine residues. Human serum albumin minimally-modified with methylglyoxal (MG_min-HSA) was bound by cell surface receptors of human monocytic THP-1 cells in vitro at 4°C: the binding constant K_d value was 377±35 nM and the number of receptors per cell was 5.9±0.2×10⁵ (n=12). N_α-Acetyl-N_δ-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine displaced MG_min-HSA from THP-1 cells, suggesting that the N_δ-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine residue was the receptor recognition factor. At 37°C, MG_min-HSA was internalised by THP-1 cells and degraded. Similar binding and degradation of human serum albumin modified by glucose-derived AGE was found but only when highly modified. MG_min-HSA, therefore, is the first example of a protein minimally-modified by AGE-like compounds that binds specifically to monocyte receptors. The irreversible modification of proteins by methylglyoxal is a potent signal for the degradation of proteins by monocytic cells in which the arginine derivative, N_δ-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine, is the receptor recognition factor. This factor is not present in glucose-modified proteins.     

First in-gel detection and purification of human xylosyltransferase II

Human xylosyltransferases I and II (XylT-I, XylT-II) are key enzymes in glycosaminoglycan biosynthesis. Knowledge about the in vivo molecular weight, oligomeric state or turnover number are essential characteristics which have been addressed in this study. XylT-II was purified from Pichia pastoris by fractionated ammonium sulfate precipitation, heparin affinity and ion exchange chromatography. XylT-II was purified over 7000-fold with a final yield of 2.6%. By utilizing mass spectra analysis we can prove its first in-gel detection showing a migration pattern behavior that confirms its in silico molecular weight of 95.8 kDa. We could determine a turnover number of 2.18 min⁻¹ or one transferred xylose molecule per one XylT-II molecule each 27.5 s. The k_cat/K_M ratio was 0.357 min⁻¹ μM⁻¹ for XylT-II using the bikunin-homologous acceptor Bio-QEEEGSGGGQKK-F. The comparison to XylT-I derived from the same organism revealed a 2.4-fold higher catalytic efficiency (0.870 min⁻¹ μM⁻¹) for XylT-I.     

Theory and Simulation of Water Permeation in Aquaporin-1

We discuss the difference between osmotic permeability p_f and diffusion permeability p_d of single-file water channels and demonstrate that the p_f/p_d ratio corresponds to the number of effective steps a water molecule needs to take to permeate a channel. While p_d can be directly obtained from equilibrium molecular dynamics simulations, p_f can be best determined from simulations in which a chemical potential difference of water has been established on the two sides of the channel. In light of this, we suggest a method to induce in molecular dynamics simulations a hydrostatic pressure difference across the membrane, from which p_f can be measured. Simulations using this method are performed on aquaporin-1 channels in a lipid bilayer, resulting in a calculated p_f of 7.1 × 10⁻¹⁴ cm³/s, which is in close agreement with observation. Using a previously determined p_d value, we conclude that p_f/p_d for aquaporin-1 measures ~12. This number is explained in terms of channel architecture and conduction mechanism.     

The interaction of nucleotides with F1-ATPase inactivated with 4-chloro-7-nitrobenzofurazan

In common with the F₁-ATPase from other sources, yeast mitochondrial F₁-ATPase was inhibited by 4-chloro-7-nitrobenzofurazan. Total inhibition of the F₁-ATPase activity was compatible with the modification of a single tyrosine residue per F₁-ATPase molecule. Radioactive labelling experiments localized this modification on a β-subunit. The inactive modified enzyme retained the capacity to bind the photoaffinity label 8-azido-1,N⁶-etheno-ATP, which has previously been shown to bind nucleotide sites of low affinity. As well, the inactive modified enzyme bound MgATP with high affinity, yielding a K_d of 14 μM. The results are consistent with the hypothesis of alternating, or cooperative, site catalysis by F₁-ATPase.     

Composition of the immune system of allophenic mice.

Mice were immunized with antigen (Rabbit Fab' fragments) attached to syngeneic, or f₁ (semi-syngeneic) irradiated spleen cells. Specific anti-rabbit Fab' plaque forming cell numbers showed that the response towards antigen on syngeneic or F₁ cells, was significantly lower than that towards the same antigen on allogeneic cells. By subsequent in vitro incubation of immune spleen cells with antigen followed by plaque assay, it was found that those spleen cells exhibiting lowered plaque forming cell numbers initially, (i.e., those mice immunized with antigen on syngeneic or F₁ cell surfaces) showed, after incubation, a response equal to or greater than those cells which initially (before in vitro incubation) demonstrated a larger response (i.e. those mice immunized with antigen on allogeneic cells).     

Antibody-catalyzed decarboxylation and aldol reactions using a primary amine molecule as a functionalized small nonprotein component

Catalytic antibody 27C1 bears binding sites for both a substrate- and a functionalized small nonprotein component in the active site. We investigated the possibility of exploiting imine and enamine intermediates using a primary amine molecule into the active site of antibody 27C1. The antibody catalyzed β-keto acid decarboxylation with a rate enhancement (k_cat/K_m/k_uncat) of 140,000, as well as highly regioselective cross-aldol reactions of ketones and p-nitrobenzaldehyde. These studies provide new strategies for the generation of catalytic antibodies possessing binding sites for functionalized components.     

Radioactive gold cluster immunoconjugates: Potential agents for cancer therapy

An 11 gold atom (undecagold) cluster was covalently attached to specific sites on Fab′, F(ab′)₂ and whole IgG molecules such that each carried 11–33 gold atoms without significant loss of native immunospecificity. Gold cluster labeled 17-1A monoclonal F(ab′)₂ antibody fragments showed 80% immunoreactivity compared to native antibody fragments in binding to human colon carcinoma cells in vitro. Radioactive gold in vivo biodistributions in nude mice with human tumors are also reported. By using clusters, potentially a larger destructive payload can be carried per antibody.     

Characteristics of β-adrenergic-agonist binding to rat adipocyte membranes: Evidence that (±)-[3H]hydroxybenzylisoproterenol interacts selectively with the adipocyte β-adrenergic receptors

The binding characteristics of the β-adrenergic agonist $\text{(±)-[}{}^3\text{H]hydroxybenzylisoproterenol}$ to rat adipocyte membranes were studied. Binding was rapid, reaching equilibrium within 10 min at 37°C (second order rate constant k₁=1.37·10⁷·M^?1·min^?1). Dissociation of specific binding by 0.5 mM (?)-isoproterenol suggested dissociation from two different sites with respective dissociation rate constants k₂ of 0.106·min^?1 and 0.011·min^?1.[³H]Hydroxybenzylisoproterenol binding was saturable (B_max=690±107 fmol/mg protein), yielding curvilinear Scatchard plots. Computer modeling of these data were consistent with the existence of two classes of [³H]hydroxybenzylisoproterenol binding sites, one having high affinity (K_D=3.5±0.7 nM) but low binding capacity (10% of the total sites) and one haveing low affinity (K_D=101±20 nM) but high binding capacity (90% of the sites). Adrenergic ligands competed with [³H]hydroxybenzylisoproterenol binding with the following order of potency=(?)-propranolol>(?)-isoproterenol>(?)-norepinephrine≈ (?)-epinephrine>>(+)-isoproterenol=(+)-propranolo, which is consistent with binding to β₁-adrenergic receptors. Competition curves of [³H]hydroxybenzylisoproterenol binding by the β-agonist (?)-isoproterenol were shallow and modeled to two affinity states of binding, whereas, competition curves by β-antagonist (?)-propranolol were steeper with Hill number near to one. Gpp[NH]p severely reduced [³H]hydroxybenzyl-isoproterenol binding, an effect which apparently resulted from the reduction of the number of both the high and low affinity sites. In membranes which had been previously exposed to (?)-isoproterenol, then number of [³H]hydroxybenzylisoproterenol binding sites was reduced by 50%, an effect which apparently resulted from the loss of part of both the high and low affinity state binding sites. Finally, the ability of (?)-isoproterenol to stimulate adenylate cyclase correlate closely with the ability of (?)-isoproterenol to displace [³H]hydroxybenzylisoproterenol binding. Comparison of these findings with the binding characteristics of the β-antagonist [³H]dihydroalprenolol to rat adipocyte membranes, led to conclude that [³H]hydroxybenzylisoproterenol can be successfully used to label the β-adrenergic receptors of rat fat cells and suggests that it might be a better ligand than [³H]dihydroalprenolol in these cells.     

Antibody coated gold particles containing radioactive gold in the demonstration of cell surface molecules

Summary Gold particles of varying size which contain either ¹⁹⁵Au or ¹⁹⁸Au were prepared using white phosphorus or sodium citrate as the reducting agent. After coating with specific antibody to blood group A antigen or human IgG these particles were used to determine the number of particles binding to the surface of A₁ RBC's or rat RBC's to which human IgG had been attached. The number of particles binding to the surface of cells correlated with the number of antibody coated gold particles in the fluid bathing the cells as well as the number of antigen molecules on the cell surface. That is, the number of particles binding increased as the particle density of the suspension increased and as the cell surface antigen density increased. Under the conditions of the experiments, both blood group A antigen and human IgG appeared to be randomly distributed over the surface of the cells in TEM and SEM preparations. This approach permitted the quantitation of the number of gold particles bound per cell and at the same time, the examination of the distribution of the particles over the surface of the same cell population by TEM and SEM.     

The radial glia antibody RC2 recognizes a protein encoded by Nestin

The RC2 antibody is widely used to label mouse radial glial cells in the developing central nervous system. While the antibody is known to recognize a 295-kDa intermediate filament proximal protein, the gene encoding the RC2 antigen remains to be identified. Here, we present evidences clearly demonstrating that Nestin encodes the RC2 antigen. First, the RC2 antigen and nestin have the same molecular weight and very similar tissue distribution. Second, genetic manipulations altering nestin expression also exert the same effect on the expression of the RC2 antigen. In particular, Nestin null mutation completely abolishes the RC2 immunoreactivity. Third, the expression of a truncated mouse nestin in Nestin−/− cells produces a small RC2 antigen whose size is the same to that of the truncated nestin. Furthermore, our data suggest that the RC2 antibody recognizes the C-terminal domain of nestin with unidentified posttranslational modification(s).     

Isolation and chemical characterization of a melanoma-associated proteoglycan antigen

Many melanoma-associated antigens have been identified by monoclonal antibodies. One of these monoclonal antibodies, O₁-94-45, binds only to melanomas, nevus cells, some astrocytomas, and fetal epitheloid cells. There are approximately 100,000 cell surface antigens per melanoma cell with an association constant of 3 × 10⁸m^?1. The antigen is efficiently extracted from the membrane only in the presence of detergent and is, therefore, bound by hydrophobic forces. However, it is also shed into the culture supernatant during normal cell growth. The two components of the O₁-95-45 antigen are a chondroitin sulfate proteoglycan (CSP, >500,000 Da) and a glycoprotein gp260 (260,000 Da, pI 6.9). CSP contains chondroitin sulfate and N-linked and O-linked oligosaccharides. Only N-linked saccharides were associated with gp260. The antigenic site is expressed on both components and is heat-sensitive. Since the CSP was converted to gp260 by chondroitinase, the protein cores of the two molecules are the same or similar. For more detailed study the O₁-95-45 antigen was purified by immunoaffinity chromatography. The amino acid composition of the purified antigen was relatively polar with an unusually high Leu content and low Lys content. Initial attempts to sequence the antigen were unsuccessful probably due to a blocked N-terminus. CSP and gp260 were partially separated by gel filtration chromatography, and both were found to carry the O₁-95-45 antigenic determinant. Three other monoclonal antibodies were found to bind the purified antigen at a site or sites different from the O₁-95-45 epitope and one other monoclonal antibody may bind at the same site. Two of these antibodies were used for a double determinant immunoassay.