Uptake of methotrexate linked to an anti-EL4-lymphoma antibody by EL4 cells

Summary To study the mechanism of tumor inhibition, the uptake of methotrexate (MTX) covalently linked to a rabbit IgG antibody against a tumor-associated antigen on the surface of mouse EL4 lymphoma cells (AELG) has been compared with the uptake of free MTX and of MTX covalently linked to normal rabbit IgG (NRG). When EL4 cells were incubated at 37°C with 10 M free MTX uptake leveled off after 30 min, at 30 pmol/mg protein. In contrast, uptake of both conjugates under these conditions continued throughout an observation period of 6 h. At 6 h the net uptake of MTX bound to AELG was 40 pmol/mg protein and that of MTX bound to NRG was 24 pmol/mg protein. These results show that both MTX-AELG and MTX-NRG conjugates are taken up by EL4 cells. The rate at which EL4 cells took up bound MTX was much slower than that of free MTX but, at 6 h, the net uptake of MTX-AELG exceeded that of the free drug. Abbreviations used in this paper: AELG, antiEL4 IgG; NRG, normal rabbit IgG; MTX, methotrexate; PBS, 0.01 M sodium phosphate, pH 7.1, containing 0.145 M sodium chloride     

Synthesis of site-specific methotrexate-IgG conjugates

Summary With a view to increasing drug incorporation without loss of antibody activity, tritium-labeled methotrexate (MTX) was covalently linked to a polyclonal rabbit IgG antibody against bovine serum albumin and a monoclonal mouse IgG antibody against human renal cancer (Dal K20) by a site-specific method based on hydrazone bond formation between MTX hydrazide and the aldehyde groups generated by periodate oxidation of carbohydrate moieties in IgG (which are uncommon in the antigen-binding region). These conjugates were compared with the corresponding non-site-specific MTX-IgG conjugates produced by the N-hydroxysuccinimide active-ester method with regard to synthesis, stability, retention of antibody activity, inhibition of the target enzyme dihydrofolate reductase and antitumor effect. Incorporation levels achieved with the hydrazide method were no greater than with the active-ester method, typically 6–7 mol MTX/mol IgG. Approximately the same dihydrofolate-reductase-inhibitory capacity was observed for MTX bound by either method. Hydrazide conjugates lost bound drug more rapidly than active-ester conjugates on freezing and thawing, on incubation at 37° C and 51° C, and in the presence of serum or rat liver homogenates. Exposure to rat liver homogenates at 37° C, pH 4.6, for 24 h led to the loss of 50%–60% of the bound drug from hydrazide conjugates compared to 20%–30% from the active ester conjugates. Bio-Gel P-2 chromatography of low-molecular-mass fractions, obtained after exposure of each of the conjugates to liver homogenates, revealed the presence of a compound that had the same elution volume and R _F on thin-layer chromatography as free MTX. Enzyme-linked immunosorbent assay showed loss of antibody activity of both types of conjugates at 51° C and on freezing and thawing. In a clonogenic assay, the active-ester conjugate of Dal K20 appeared to be equally effective or slightly better as a tumor inhibitor than the corresponding hydrazide conjugate. The hydrazide method may be useful in linking MTX to those monoclonal antibodies that tend to denature when subjected to the active-ester method of linkage. Abbreviations used: aBSA, rabbit anti-(bovine serum albumin) IgG; EDCI, 3-ethyl-1-(3-dimethylaminopropyl)carbodiimide; ELISA, enzyme-linked immunosorbant assay; IC₅₀, concentration giving 50% inhibition; MTX, methotrexate; MTXAE, N-hydroxy-succinimide-based active ester of MTX; MTXAE-IgG, MTX-IgG conjugate prepared by the active-ester method; MTXH, methotrexate hydrazide; MTXH-IgG, MTX-IgG conjugate prepared by the hydrazide method; PBS, 0.01 M sodium phosphate, pH 7.1, containing 0.145 M sodium chloride; TLC, thin-layer chromatography     

A monoclonal antibody against hinge-cleaved IgG restores effector function to proteolytically-inactivated IgGs in vitro and in vivo

We report a chimeric monoclonal antibody (mAb) directed to a neo-epitope that is exposed in the IgG lower hinge following proteolytic cleavage. The mAb, designated 2095–2, displays specificity for IdeS-generated F(ab’)₂ fragments, but not for full-length IgG or for closely-related F(ab’)₂ fragments generated with other proteases. A critical component of the specificity is provided by the C-terminal amino acid of the epitope corresponding to gly-236 in the IgG1 (also IgG4) hinge. By its ability to bind to IdeS-cleaved anti-CD20 mAb, mAb 2095–2 fully restored antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against WIL2-S cells to the otherwise inactive anti-CD20 IgG1 F(ab’)₂ fragment. Similarly, 2095–2 reinstated ADCC against MDA-MB-231 cells to an anti-CD142 IgG1 F(ab’)₂ fragment. mAb 2095–2 was also capable of eliciting both CDC and ADCC to IgG4 F(ab’)₂ fragments, an IgG subclass that has weaker ADCC and CDC when intact relative to intact IgG1. The in vitro cell-based efficacy of 2095–2 was extended to the in vivo setting using platelets as a cell clearance surrogate. In a canine model, the co-administration of 2095–2 together with IdeS-generated, platelet-targeting anti-CD41/61 F(ab’)₂ fragment not only restored platelet clearance, but did so at a rate and extent of clearance that exceeded that of intact anti-CD41/61 IgG at comparable concentrations. To further explore this unexpected amplification effect, we conducted a rat study in which 2095–2 was administered at a series of doses in combination with a fixed dose of anti-CD41/61 F(ab’)₂ fragments. Again, the combination, at ratios as low as 1:10 (w/w) 2095–2 to F(ab’)₂, proved more effective than the anti-CD41/61 IgG1 alone. These findings suggest a novel mechanism for enhancing antibody-mediated cell-killing effector functions with potential applications in pathologic settings such as tumors and acute infections where protease activity is abundant.     

Low-dose methotrexate enhances cycling of highly anaplastic cancer cells

We previously showed that cellular RedOx state governs the G₁-S transition of AH130 hepatoma, a tumor spontaneously reprogrammed to the embryonic stem cell stage. This transition is impaired when the mithocondrial electron transport system is blocked by specific inhibitors (antimycin A) or the respiratory chain is saturated by adding to the cells high concentrations of pyruvate. The antimycin A or pyruvate block is removed by the addition of adequate concentrations of folate (F). This suggests that the G₁-S transition of AH130 cells depends on a respiration-linked step of DNA synthesis related to folate metabolism. In the study reported here, we characterized the effects of methotrexate (MTX), an inhibitor of dihydofolate-reductase, on the G₁-S transition of hepatoma cells, in the absence or the presence of exogenously added F, dihydrofolate (FH₂) or tetrahydrofolate (FH₄). MTX, at 1 μM or higher concentrations, inhibited G₁-S transition. This inhibition was completely removed by exogenous folates. Surprisingly, 10 nM MTX stimulated G₁-S transition. The addition of F, but not FH₂ or FH₄, significantly increased this effect. Furthermore, 10 nM MTX removed the block of the G₁-S transition operated by antimycin A or pyruvate, an effect which was enhanced in the presence of F. Finally, the stimulatory effect of 10 nM MTX was inhibited in the presence of serine. Our findings indicated that, under certain conditions, MTX may stimulate, rather than inhibiting, the cycling of cancer cells exhibiting a stem cell-like phenotype, such as AH130 cells. This may impact the therapeutic use of MTX and of folates as supportive care.     

Fragments of human γG immunoglobulins produced by a porcine pancreatic esterase

The effect of a porcine pancreatic esteroproteolytic enzyme on human IgG has been described. A sequential breakdown of the molecule occurs. The first cleavage results in the formation of an F′c fragment together with an F(ab)₂ fragment. A subsequent proteolysis of the F(ab)₂ fragment liberates two Fab fragments. Each fragment has been characterized by its antigenic properties, molecular weight, and sulfhydryl content.     

Influence of the bispecific antibody IgG subclass on T cell redirection

ABSTRACT

T cell redirection mediated by bispecific antibodies (BsAbs) is a promising cancer therapy. Dual antigen binding is necessary for potent T cell redirection and is influenced by the structural characteristics of a BsAb, which are dependent on its IgG subclass. In this study, model BsAbs targeting CD19xCD3 were generated in variants of IgG1, IgG2, and IgG4 carrying Fc mutations that reduce FcγR interaction, and two chimeric IgG subclasses termed IgG1:2 and IgG4:2, in which the IgG1- or IgG4-F(ab)₂ are grafted on an IgG2 Fc. Molecules containing an IgG2 or IgG4-F(ab)₂ domain were confirmed to be the most structurally compact molecules. All BsAbs were shown to bind both of their target proteins (and corresponding cells) equally well. However, CD19xCD3 IgG2 did not bind both antigens simultaneously as measured by the absence of cellular clustering of T cells with target cells. This translated to a reduced potency of IgG2 BsAbs in T-cell redirection assays. The activity of IgG2 BsAbs was fully restored in the chimeric subclasses IgG4:2 and IgG1:2. This confirmed the major contribution of the F(ab)₂ region to the BsAb’s functional activity and demonstrated that function of BsAbs can be modulated by engineering molecules combining different Fc and F(ab)₂ domains.

Abbreviations: ADCC: Antibody-dependent cellular cytotoxicity; AlphaScreen^TM: Amplified Luminescent Proximity Homogeneous Assay Screening; ANOVA: Analysis of variance; BiTE: bispecific T-cell engager; BSA: bovine serum albumin; BsAb: bispecific antibody; cFAE: controlled Fab-arm exchange; CDC: complement-dependent cellular cytotoxicity; CIEX: cation-exchange; CIR: chimeric immune receptor; DPBS: Dulbecco’s phosphate-buffered saline; EC₅₀ value: effective concentration to reach half-maximum effect; EGFR: epidermal growth factor receptor; EI: expansion index (RA_t=x/RA_t=0); FACS: fluorescence-activated cell sorting; FVD: fixable viability dye; HI-HPLC: hydrophobic interaction HPLC; HI-FBS: heat-inactivated fetal bovine serum; HPLC: high-pressure liquid chromatography; IC₅₀ value: effective concentration to reach half-maximum inhibition; IQ: Inhibition Quotient; IS: immunological synapse; MES: 2-(N-morpholino)ethanesulfonic acid; R-PE: recombinant phycoerythrin; RA: red area in μm²/well; RD: receptor density; RFP: red fluorescent protein; R_g: radius of gyration; RSV: respiratory syncytial virus; SAXS: small-angle x-ray scattering; scFv: single-chain variable fragment; SD: standard deviation; SPR: surface plasmon resonance; WT: wild-type     

Regression of human melanoma xenografts in nude mice injected with methotrexate linked to monoclonal antibody 225.28 to human high molecular weight-melanoma associated antigen

Summary Intravenous injections into nude mice of 5 mg/kg methotrexate (MTX) linked to the antibody to human high molecular weight-melanoma associated antigen (HMW-MAA), monoclonal antibody (mAb) 225.28, an IgG_2a, on days 1, 4, 7, 10 and 14, starting 24 h after subcutaneous inoculation of 2 × 10⁶ cultured human M21 melanoma cells inhibited mean tumor volume by 90% on day 14 and by 65% on day 50 after the beginning of the treatment. Injections of equimolar amounts of free MTX and MTX linked to normal mouse IgG or to an isotypematched myeloma protein did not inhibit tumor growth significantly. MTX linked to mAb 225.28 did not inhibit the xenograft of a subline of human melanoma cell line M21 without detectable expression of HMW-MAA. In a clonogenic assay, the MTX-225.28 conjugate was three times more potent in inhibiting the growth of M21 melanoma cells than free MTX, but did not inhibit the growth of kidney carcinoma cells Caki-1, which do not express high-M _r MAA. In contrast, MTX linked to the mAb DAL K29, reacting with kidney carcinoma cells Caki-1, inhibited their growth but did not affect that of melanoma cells. M21 melanoma cells isolated from the residual tumor of a mouse treated with the MTX-225.28 conjugate did not differ in their reactivity with mAb 225.28 and in their sensitivity to MTX when compared with M21 cells from an untreated mouse.     

Cytotoxicities of two disulfide-bond-linked conjugates of methotrexate with monoclonal anti-MM46 antibody

Summary In studies on (antitumor antibody)-drug conjugates as potential antitumor agents, the amide derivatives of methotrexate (MTX) with cysteine and with 2-mercaptoethylamine (cysteamine) (MTX-Cys and MTX-MEA, respectively) were linked via a disulfide bond with a monoclonal antibody (MM46) to a mouse mammary tumor MM46 with attached 3-(2-pyridyldithio) propionyl groups to give conjugates of MTX with MM46 (MTX-Cys-SS-MM46 and MTX-MEA-SS-MM46, respectively). These two conjugates are both linked by a disulfide bond and are very similar in structure, but MTX-MEA-SS-MM46 showed only weak in vitro cytotoxicity against MM46 cells, whereas MTX-Cys-SS-MM46 had strong cytotoxicity. The cytotoxicity of the latter was comparable to that of the conventional direct MTX-MM46 conjugate prepared with an MTX-active ester. However, this conjugate had a greater selectivity than that of the direct conjugate, calculated as the IC₅₀ (concentration of a conjugate by MTX equivalence required for suppression of the number of viable MM46 cells to 50% of that of the untreated control) for the corresponding nonspecific conjugate divided by the IC₅₀ for the MM46 conjugate. The inhibitory activities of MTX-Cys and MTX-MEA on dihydrofolate reductase were similar. The cytotoxicity of MTX-Cys-SS-MM46 was not affected by thiamine pyrophosphate, an inhibitor of the active transport of MTX across the cell membrane, but was decreased significantly by ammonium chloride, a lysosomotropic amine. However, the cytotoxicity was decreased only to a small extent by leupeptin, an inhibitor of lysosomal cysteine proteases cathepsins B, H, and L. These results suggest that the cytotoxicity is mediated by lysosomes, and may involve lysosomal enzymes other than cathepsins B, H, and L.     

Evading pre-existing anti-hinge antibody binding by hinge engineering

Antigen-binding fragments (Fab) and F(ab′)₂ antibodies serve as alternative formats to full-length anti-bodies in therapeutic and immune assays. They provide the advantage of small size, short serum half-life, and lack of effector function. Several proteases associated with invasive diseases are known to cleave antibodies in the hinge-region, and this results in anti-hinge antibodies (AHA) toward the neoepitopes. The AHA can act as surrogate Fc and reintroduce the properties of the Fc that are otherwise lacking in antibody fragments. While this response is desired during the natural process of fighting disease, it is commonly unwanted for therapeutic antibody fragments. In our study, we identify a truncation in the lower hinge region of the antibody that maintains efficient proteolytic cleavage by IdeS protease. The resulting neoepitope at the F(ab′)₂ C-terminus does not have detectable binding of pre-existing AHA, providing a practical route to produce F(ab′)₂ in vitro by proteolytic digestion when the binding of pre-existing AHA is undesired. We extend our studies to the upper hinge region of the antibody and provide a detailed analysis of the contribution of C-terminal residues of the upper hinge of human IgG1, IgG2 and IgG4 to pre-existing AHA reactivity in human serum. While no pre-existing antibodies are observed toward the Fab of IgG2 and IgG4 isotype, a significant response is observed toward most residues of the upper hinge of human IgG1. We identify a T₂₂₅L variant and the natural C-terminal D₂₂₁ as solutions with minimal serum reactivity. Our work now enables the production of Fab and F(ab′)₂ for therapeutic and diagnostic immune assays that have minimal reactivity toward pre-existing AHA.     

Reduced intracellular content of methotrexate in an isolated MTX-resistant cell line of Nicotiana plumbaginifolia

Summary In plant cells methotrexate (MTX) may exert its toxic effect through several mechanisms, including inhibition of its target protein dihydrofolate reductase. Resistance based on a mechanism operating before MTX binds to proteins should confer protection to plant cells. A methotrexate-resistant cell line of Nicotiana plumbaginifolia was isolated by a stepwise selection procedure. This cell line survived in the presence of 10 M MTX which is 50–100 fold higher than the lethal dose for the wild type cells. Neither alteration in kinetic characteristics of dihydrofolate reductase, nor elevated binding capacity of ³H-MTX to target protein(s), were observed. However, in comparison with wild type cells, markedly lower amounts of intracellular ³H-MTX were found after the selected cell line was incubated with ³H-MTX, indicating that either reduced uptake or enhanced efflux of MTX is the major reason for MTX-resistance in this cell line.     

Development of a fluorescently labeled thermostable DHFR for studying conformational changes associated with inhibitor binding

A fluorescently-labeled, conformationally-sensitive Bacillus stearothermophilus (Bs) dihydrofolate reductase (DHFR) (C73A/S131C_MDCC DHFR) was developed and used to investigate kinetics and protein conformational motions associated with methotrexate (MTX) binding. This construct bears a covalently-attached fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC) attached at a distal cysteine, introduced by mutagenesis. The probe is sensitive to the local molecular environment, reporting on changes in the protein structure associated with ligand binding. Intrinsic tryptophan fluorescence of the unlabeled Bs DHFR construct (C73A/S131C DHFR) also showed changes upon MTX association. Stopped-flow analysis of all data can be understood by invoking the presence of two native state DHFR conformers that bind to MTX at different rates (20.2 and 0.067 μM⁻¹ s⁻¹), similar to previously published findings for Escherichia coli DHFR. Probe fluorescence of C73A/S131C_MDCC DHFR predominantly reports on MTX binding to one of the conformers while intrinsic tryptophan fluorescence of C73A/S131C DHFR reports on binding to the other conformer. This study demonstrates the use of an extrinsic fluorophore attached to a distal region to investigate ligand binding interactions that are not experimentally accessible via intrinsic tryptophan fluorescence alone. The thermostability of C73A/S131C_MDCC DHFR provides an important new tool with applications for investigating the temperature dependence of DHFR conformational changes associated with binding and catalysis.     

Mechanisms of stable serum resistance of Neisseria gonorrhoeae

Neisseria gonorrhoeae that resist complement-dependent killing by normal human serum (NHS) are sometimes killed by immune convalescent sera from patients recovering from disseminated gonococcal infection (DGI). In these studies, killing by immune serum was prevented or blocked by immunoglobulin G (IgG) or F(ab)₂ isolated from NHS. Purified human IgG antibodies directed against gonococcal protein III, contained most of the blocking activity in IgG. In addition, immune convalescent DGI serum, which did not exhibit bactericidal activity, was restored to killing by selective immunodepletion of protein III antibodies. Blocking IgG or F(ab)₂ prepared from IgG, partially inhibited binding of bactericidal antibody to N. gonorrhoeae. Also, binding of a monoclonal antibody recognizing N. gonorrhoeae outer membrane protein PIII was almost completely inhibited by blocking F(ab)₂.Presensitization of N. gonorrhoeae with increasing concentrations of blocking IgG or F(ab)₂ before incubation with bactericidal antibody and an antibody free source of complement, increased consumption and deposition of the third component of human complement (C3) and the ninth component of complement (C9) but inhibited killing in dose-related fashion.     

Effect of antibody to detergent solubilized zonae pellucidae on in vivo and in vitro fertilization in the mouse

An antiserum was produced in one rabbit against mouse zonae pellucidae solubilized with 70 mM Na₂SO₃, 1% SDS, and 0.04 mM CuSO₄. An IgG of zona antibody completely inhibited fertilization both in vitro and in vivo in the mouse. F(ab′)₂ fragment obtained by pepsin digestion of IgG zona antibody inhibited fertilizability of eggs in vitro but did not inhibit fertilization in vivo after passive immunization.     

Adsorption of IgG onto hydrophobic teflon. Differences between the F(ab) and F(c) domains

The effect of differences in the degree of hydrophobicity of protein patches/fragments on the adsorption behaviour of the protein is investigated. The adsorption isotherm of a monoclonal mouse anti-human immunoglobulin G (isotype 2b) onto hydrophobic Teflon particles is measured using a depletion method. The adsorption-induced denaturation of the immunoglobulin as a function of the adsorbed amount is studied by differential scanning calorimetry, and the corresponding rearrangements in the secondary structure of the whole IgG molecule and its F(ab) and F(c) fragments are determined by circular dichroism spectroscopy. The effects of adsorption on the F(ab) and F(c) fragments in the intact IgG molecule occur independently. Adsorption of the whole IgG molecule leads to denaturation of the F(ab) fragments, whereas the F(c) fragment remains unperturbed; adsorption of the isolated fragments results in structural changes in both F(ab) and F(c). The surface hydrophobicity of the isolated fragments was studied by HPLC. These experiments support the hypothesis that differences in the degree of denaturation between F(ab) and F(c) are due to the higher degree of hydrophobicity of the F(ab) fragment. The adsorption-induced changes in the secondary structure are more prominent for the isolated fragments as compared to intact IgG. This is ascribed to the higher flexibility of the isolated fragment, as compared to the fragment in the whole molecule.     

The Effect of Molecular Weight,PK, and Valency on Tumor Biodistribution and Efficacy of Antibody-Based Drugs

Poor drug delivery and penetration of antibody-mediated therapies pose significant obstacles to effective treatment of solid tumors. This study explored the role of pharmacokinetics, valency, and molecular weight in maximizing drug delivery. Biodistribution of a fibroblast growth factor receptor 4 (FGFR4) targeting CovX-body (an FGFR4-binding peptide covalently linked to a nontargeting IgG scaffold; 150 kDa) and enzymatically generated FGFR4 targeting F(ab)₂ (100 kDa) and Fab (50 kDa) fragments was measured. Peak tumor levels were achieved in 1 to 2 hours for Fab and F(ab)₂versus 8 hours for IgG, and the percentage injected dose in tumors was 0.45%, 0.5%, and 2.5%, respectively, compared to 0.3%, 2%, and 6% of their nontargeting controls. To explore the contribution of multivalent binding, homodimeric peptides were conjugated to the different sized scaffolds, creating FGFR4 targeting IgG and F(ab)₂ with four peptides and Fab with two peptides. Increased valency resulted in an increase in cell surface binding of the bivalent constructs. There was an inverse relationship between valency and intratumoral drug concentration, consistent with targeted consumption. Immunohistochemical analysis demonstrated increased size and increased cell binding decreased tumor penetration. The binding site barrier hypothesis suggests that limited tumor penetration, as a result of high-affinity binding, could result in decreased efficacy. In our studies, increased target binding translated into superior efficacy of the IgG instead, because of superior inhibition of FGFR4 proliferation pathways and dosing through the binding site barrier. Increasing valency is therefore an effective way to increase the efficacy of antibody-based drugs.     

Characteristics of methotrexate polyglutamate formation in cultured hepatic cells

Upon exposure of primary monolayer cultures of hepatocytes and H35 hepatoma cells, methptrexate (MTX) is taken up by carrier-mediated mechanisms and converted to γ-glutamyl derivatives with one to four residues being added. Under conditions that result in 90% or greater conversion, the primary metabolite in both cell types is MTX with three additional glutamates (4-NH₂-10-CH₃PteGlu₄). When the time-dependent synthesis of MTX polyglutamates (4-NH₂-10-CH₃PteGlu₂ and higher) at extracellular concentrations of 10 and 100 μm methotrexate is measured, both cell types exhibit linear synthesis for 4 to 6 hr, at which time an apparent steady state intracellular concentration of approximately 40 μm is reached. The concentration of MTX polyglutamate synthesized is not due a restriction in MTX since the hepatocytes and H35 cells accumulated 400 and 138 μm intracellular methotrexate, respectively, after 24 h in the presence of 100 μm extracellular MTX. Examination of MTX polyglutamate formation following a 24-h incubation showed concentration dependence with respect to intra- and extracellular MTX. Saturation was reached at a medium concentration of approximately 2 μm with both cell types which corresponded to 10 to 12 μm intracellular MTX. Placement of cells at steady state in medium lacking MTX results in the rapid equilibration of all free intracellular MTX with the medium. The MTX polyglutamates leave the cell by a slow loss of intact polyglutamates and also by intracellular cleavage to MTX followed by efflux. The longer-chain-length γ-glutamyl derivatives (Glu_4–5) are more avidly retained by the cells than the shorter ones (Glu_2–3).     

Phosphate uptake in the yeast Candida tropicalis: purification of phosphate-binding protein and investigations about its role in phosphate uptake

The purification of a phosphate-binding protein (P_iBP₂) by immunoadsorption is described. The entire anti phosphate-binding protein 2 antibodies as well as the Fab fragments obtained from these antibodies inhibit P_i uptake by whole cells. The inhibition is a mixed type of inhibition (V _m and K _m are affected). These results should be regarded as a possible involvement of phosphate-binding protein 2 in P_i uptake. The binding of ¹²⁵I-labelled fragments prepared from anti phosphate-binding protein 2 antibodies to whole cells, to shocked cells and to protoplasts has been investigated. The results confirm the release of phosphate-binding protein by osmotic shock and during protoplast formation. From these findings, a cell-wall localisation, near the cell surface of the phosphate-binding protein should be proposed.Abbreviations P_i inorganic phosphate - P_iBP phosphate-binding protein - Tris Tris (hydroxymethyl)-aminoethane - MES (2(N-Morpholino) ethanesulfonic acid - BSA bovine serum albumin - EDTA ethylene diamine tetraacetic acid, disodium salt - PMSF phenylmethyl sulfonyl fluoride - SDS sodium dodecyl sulfate - Fab fragments, fragment antigen binding     

Targeting of the antiviral protein from Phytolaccaamericana with an antibody

The antiviral protein (PAP) of $\text{Phytolacca}\text{americana}$ was conjugated with the Fab' fragment of IgG from a rabbit antiserum against murine leukemia L1210 cells via a disulfide bond employing $\text{N}$-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) as the coupling agent. The conjugate showed a potent $\text{in}\text{vitro}$ cytotoxicity against L1210 cells which was competitively blocked by F(ab′)₂ directed against L1210 cells. PAP itself did not exhibit the cytotoxicity at the concentration corresponding to the PAP content in the conjugate concurrently tested.     

Transient inhibition of DNA synthesis by 5-fluorodeoxyuridine leads to overexpression of dihydrofolate reductase with increased frequency of methotrexate resistance

Transient but incomplete suppression of DNA synthesis by a single exposure of an asynchronous population of cells to 5-fluoro-2'-deoxyuridine (FdUrd) increases the frequency of appearance of methotrexate (MTX)-resistant colonies. This increase was greater than 10-fold following a 6-h incubation of cells with 3 microM FdUrd prior to selection in MTX, an interval one-half the normal L1210 cell cycle time. During this period of exposure to FdUrd, DNA synthesis decreased to 25% of control rates and cells accumulated at the G1/S interface. The 6-h incubation with FdUrd resulted in greater than a 2.5-fold increase in the dihydrofolate reductase protein level in the treated cell population, which was accounted for, at least in part, by increased de novo synthesis of the enzyme as assessed by [35S]methionine labeling. This increase in dihydrofolate reductase was associated with a decrease in growth inhibition by MTX. A brief reversal (2 h) of FdUrd-induced DNA synthesis inhibition by the addition of thymidine eliminated the amplification of dihydrofolate reductase and the enhanced emergence of MTX-resistant clones. Beyond this, an analysis of clones that survive MTX selection indicates that the dihydrofolate reductase gene copy in cells spontaneously resistant to 50 nM MTX and those which resulted after the additional pretreatment with FdUrd for 6 h are comparable with a 2-4-fold amplification of enzyme in most clones. These studies demonstrate that FdUrd enhancement of dihydrofolate reductase expression can have a profound effect upon the incidence and expression of MTX resistance and that dihydrofolate reductase gene amplification may be another basis for antagonism between these agents.     

Fc and Fab Fragments from IgG<Subscript>2</Subscript> Human Immunoglobulins Characterized

Papain digestion of 7S immunoglobulin G (IgG) produces two 3.5S Fab fragments and one 3.5S Fc fragment^1–8. The Fab fragment contains one light chain and one Fd fragment and is still able to combine specifically univalently with antigen. The Fc fragment is a dimer of the carboxyl terminal half of the heavy chain. Pepsin splits 7S IgG into some small peptides derived from Fc and one 5S F(ab′)₂ fragment, which contains both antigen-binding sites. Based on this information, some investigators^6,7 have postulated that pepsin splits the γ chains at the C-terminal side of the inter-heavy chain disulphide bridges, whereas papain splits at the N-terminal side of the inter-heavy chain disulphide bridges. We report here evidence that this model does not apply to all IgG subclasses. In the case of human IgG₂ subclass myeloma proteins, papain splits initially at the C-terminal side of inter-heavy chain disulphide bridges. We also show that the amino-acid sequence of the Fc fragment of human IgG₂ subclass so far determined has approximately 95% homology with that of human IgG₁ and IgG₄ subclasses reported by others^9–15.