HIV-infected cells are killed by rCD4-ricin A chain.

The gp120 envelope glycoprotein of the human immunodeficiency virus (HIV), which is expressed on the surface of many HIV-infected cells, binds to the cell surface molecule CD4. Soluble derivatives of recombinant CD4 (rCD4) that bind gp120 with high affinity are attractive vehicles for targeting a cytotoxic reagent to HIV-infected cells. Soluble rCD4 was conjugated to the active subunit of the toxin ricin. This conjugate killed HIV-infected H9 cells but was 1/1000 as toxic to uninfected H9 cells (which do not express gp120) and was not toxic to Daudi cells (which express major histocompatibility class II antigens, the putative natural ligand for cell surface CD4). Specific killing of infected cells can be blocked by rgp120, rCD4, or a monoclonal antibody to the gp120 binding site on CD4.

Increasing the serum persistence of an IgG fragment by random mutagenesis.

The major histocompatibility complex (MHC) class I-related receptor FcRn is involved in regulating serum gammaglobulin (IgG) levels in mice. With the aim of increasing the serum half-life of a recombinant murine Fc gamma 1 fragment, the affinity for binding to FcRn at pH 6.0 has been increased by random mutagenesis of Thr252, Thr254, and Thr256 followed by selection using bacteriophage display. These residues were chosen as they are in proximity to the FcRn-IgG (Fc) interaction site. Two mutants with higher affinity (due to lower off-rates) than the wild-type Fc have been isolated and analyzed in pharmacokinetic studies in mice. The mutant with the highest affinity has a significantly longer serum half-life than the wild type fragment, despite its lower off-rate from FcRn at pH 7.4. The results provide support for the involvement of FcRn in the homeostasis of serum IgGs in mice. The indications that a homologous FcRn regulates IgG levels in humans suggest that this approach has implications for increasing the serum persistence of therapeutic antibodies.

Anti-CD19 immunotoxin enhances the activity of chemotherapy in severe combined immunodeficient mice with human pre-B acute lymphoblastic leukemia.

The anti-CD19 immunotoxin (IT) (HD37-dgRTA) is effective in killing B-lineage leukemia cells and in curing severe combined immunodeficient mice with acute lymphoblastic leukemia. The present study aimed to identify effective combinations of HD37-dgRTA and chemotherapeutic agents. The in-vitro cytotoxicity assays demonstrate that the combination of HD37-dgRTA and either daunorubicin or vincristine is effective. The in-vivo experiments using HD37-dgRTA with vincristine prolonged the survival of mice compared to the chemotherapeutic agent or IT (90.7 vs. 147.1 days). Also, 80% of the mice treated with IT plus vincristine were long-term survivors.

Evaluation of ricin A chain-containing immunotoxins directed against CD19 and CD22 antigens on normal and malignant human B-cells as potential reagents for in vivo therapy.

Ricin A chain-containing immunotoxins (IT-As) specific for the human B-cell antigens, CD22 and CD19, were constructed using the monoclonal antibodies, HD6 and HD37, respectively. IT-As were prepared by coupling intact antibodies, F(ab')2, or Fab' fragments to native or chemically deglycosylated ricin A chain. The IT-As were then evaluated for cytotoxicity to normal and neoplastic human B-cells in vitro with the major objective of appraising their suitability for in vivo therapy of human B-cell tumors. The IT-As prepared with both the HD6 and HD37 antibodies were specifically toxic to normal B-cells and to most of the neoplastic B-cell lines tested. However, the IT-As prepared from HD6 were generally more potent than those prepared from HD37. On Daudi cells, to which the two antibodies bound in similar numbers and with similar affinities, IT-As prepared with intact HD6 antibody or its Fab' fragment were 10-fold and 1.5- to 4-fold more potent, respectively, than the corresponding HD37 IT-As. The IT-As constructed from intact HD6 antibody and native or deglycosylated A chain reduced protein synthesis in Daudi cells by 50% at a concentration of 1.2 X 10(-11) M indicating that they were only 5-fold less toxic to the cells than ricin itself. Intact HD37 IT-As produced equivalent inhibition of protein synthesis at 1.5 X 10(-10) M. With both antibodies, IT-As constructed from the Fab' fragments were 10- to 20-fold less potent than their intact antibody counterparts. Different neoplastic B-cell lines varied in sensitivity to the IT-As. In most cases, their sensitivity correlated with the levels of CD19 and CD22 antigens expressed. Neither HD6 nor HD37 IT-As affected the ability of normal human bone marrow cells to form granulocyte-macrophage colony-forming units in soft agar, suggesting that both antigens are absent from these progenitor cells. Examination of sections of frozen human tissues using immunoperoxidase staining procedures indicated that the antibodies did not bind to a panel of normal tissues lacking B-lymphocytes. These results suggest that HD6 and HD37 IT-As are candidates for in vivo therapy in humans with certain B-cell tumors. However, HD6 IT-As are more potent, reduce protein synthesis more completely, and hence appear to be the ITs of choice for treating tumors expressing the CD22 antigen.

Immunotoxins in the therapy of cancer: from bench to clinic.

This review presents only those contributions that have progressed from the bench to the clinic using murine monoclonal antibodies coupled chemically to toxins, their subunits or ribosome-inactivating proteins. The rationale and progress in the development, characterization, preclinical testing and clinical trials are discussed.

Treatment of SCID/human B cell precursor ALL with anti-CD19 and anti-CD22 immunotoxins.

The anti-CD19 (HD37-dgRTA) and anti-CD22 (RFB4-dgRTA) immunotoxins (ITs) are murine IgG(1) monoclonal antibodies (Mabs) conjugated to a deglycosylated ricin A chain (dgRTA). They are effective in killing B-lineage non-Hodgkin's lymphoma (NHL) cells in vitro, in vivo and in adult patients with B-lineage NHL. The potential of these agents for the treatment of childhood B-precursor acute lymphoblastic leukemia (ALL) is unknown. The anti-CD19 and anti-CD22 ITs should have anti-tumor activity against childhood B-lineage ALL since both target antigens are expressed on the surface of these cells. We have previously shown that, in vitro these two ITs selectively kill leukemia cells obtained from children with leukemia. To evaluate the efficacy of our ITs in an in vivo model we injected the human pre-B ALL cell line, NALM-6-UM1, into severe combined immunodeficient (SCID) mice. We tested the ability of two ITs to prolong survival or cure mice of both early and advanced tumors. In early disease, treatment with HD37-dgRTA, RFB4-dgRTA, or Combotox (an equimolar concentration of the two ITs) significantly improved their survival. In advanced disease, treatment with RFB4-dgRTA or Combotox significantly improved survival. Overall there were 10 long-term survivors who were cured, as determined by survival beyond 150 days with no evidence of disease as determined by polymerase chain reaction (PCR) analysis.

Treatment of refractory Hodgkin's lymphoma patients with an anti-CD25 ricin A-chain immunotoxin.

The anti-CD25 immunotoxin RFT5.dgA was constructed by coupling the monoclonal antibody RFT5 via a sterically hindered disulfide linker to deglycosylated ricin A-chain and was administered to patients with relapsed Hodgkin's lymphoma in four bolus infusions over 7 days (day 1, 3, 5 and 7). The maximum tolerated dose in these patients as defined in a previous phase I study was 15 mg/m2. Subsequently, further patients were enrolled at the maximum tolerated dose and a total of 18 patients were treated at this level. All patients had signs of progressive disease and were heavily pretreated. Side-effects in this trial were moderate and related to vascular leak syndrome. Five of 18 patients experienced NCI grade III toxicities including weakness, edema, dyspnea, and myalgia. Eleven of 16 (69%) patients receiving two or more cycles produced human anti-ricin antibodies and human anti-mouse antibodies (>/=1.0 microg/ml). Seventeen of 18 patients were evaluable for clinical response. These included two partial remissions. One patient demonstrated minor response and five patients stable diseases. We conclude that RFT5.dgA is of moderate clinical efficacy in this group of heavily pretreated refractory patients. Leukemia (2000) 14, 129-135.

Anti-CD19 antibodies inhibit the function of the P-gp pump in multidrug-resistant B lymphoma cells.

After chemotherapy, tumor cells with multidrug resistance (MDR) often emerge. MDR is attributable to the expression of membrane transport proteins that inhibit the cellular influx and increase the efflux of many chemotherapeutic drugs. One such protein is P-glycoprotein (P-gp), which functions as an ATP-dependent active transporter. Recently, an anti-P-gp monoclonal antibody (MAb) that inhibits P-gp has been described. Previous studies from our laboratory using the anti-CD19 B-cell lymphoma-reactive MAb, HD37, have suggested that HD37 may also influence MDR. To test this directly, we used Namalwa/MDR1 cells to study the effect of HD37 on the efflux of rhodamine 123 from these cells. We found that HD37 and three other anti-CD19 MAbs inhibited the efflux of rhodamine 123 from Namalwa/MDR1 cells with approximately 50% of the efficiency of the well-known chemosensitizer, verapamil. In contrast, MAbs against seven other molecules expressed on these cells were ineffective. The inhibitory activity of HD37 did not require an Fc portion; F(ab')2 fragments were effective, but Fab' fragments were not, suggesting that higher avidity binding and/or cross-linking of CD19 are necessary. We could find no evidence that HD37 recognizes a cross-reactive epitope on P-gp, modulates P-gp from the cell surface, or enhances the ATPase activity of membranes from treated cells.

A phase I study of combination therapy with immunotoxins IgG-HD37-deglycosylated ricin A chain (dgA) and IgG-RFB4-dgA (Combotox) in patients with refractory CD19(+), CD22(+) B cell lymphoma.

This study used an 8-day continuous infusion regimen of a 1:1 mixture of two immunotoxins (ITs) prepared from deglycosylated ricin A chain (dgA) conjugated to monoclonal antibodies directed against CD22 (RFB4-dgA) and CD19 (HD37-dgA; Combotox) in a Phase I trial involving 22 patients with refractory B cell lymphoma to determine the maximum tolerated dose, clinical pharmacology, and toxicity profile and to characterize any clinical responses. Adult patients received a continuous infusion of Combotox at 10, 20, or 30 mg/m2/192 h. No intrapatient dose escalation was permitted. Patients with > or =50 circulating tumor cells (CTCs)/mm3 in peripheral blood tolerated all doses without major toxicity. The maximum level of serum IT (Cmax) achieved in this group was 345 ng/ml of RFB4-dgA and 660 ng/ml of HD37-dgA (1005 ng/ml of Combotox). In contrast, patients without CTCs (<50/mm3) had unpredictable clinical courses that included two deaths probably related to the IT. Additionally, patients exhibited a significant potential for association between mortality and a history of either autologous bone marrow or peripheral blood stem cell transplants (P2 = 0.003) and between mortality and a history of radiation therapy (P2 = 0.036). In patients with CTCs, prior therapies appeared to have little impact on toxicity. Subsequent evaluation of the ITs revealed biochemical heterogeneity between two lots of HD37-dgA. In addition, HD37-dgA thawed at the study site tended to contain significant particulates, which were not apparent in matched controls stored at the originating site. This suggests that a tendency to aggregate may have resulted from shipping, storage, and handling of the IT that occurred prior to preparation for administration. It is not clear to what extent, if any, the aggregation of HD37-dgA IT was related to the encountered clinical toxicities; however, the potential to aggregate does suggest one possible basis for problems in our clinical experience with HD37-dgA and leads us to the conclusion that non-aggregate-forming formulations for these ITs should be pursued prior to future clinical trials.

Preparation and some properties of dimeric rabbit IgG antibody.

By reacting rabbit IgG with the fragment AB of protein A from S. aureus (mol. wt 14,000) an IgG dimer was formed with an approx. mol. wt of 320,000 and a molar composition of IgG2-AB1. A hybrid dimer with dual specificity consisting of IgG anti-sheep red blood cells/AB/IgG anti-bovine red blood cells was also obtained by reacting successively both rabbit antibodies with the AB fragment. The immunologic properties (affinity for antigen, complement activation and binding to Fc receptors) of the dimeric IgG were investigated in comparison with monomeric rabbit IgG and a tetrameric IgG obtained by reaction with protein A, (IgG2-protein A1)2.

Specific cytotoxic activity of normal T-lymphocytes coated with multivalent hybrid antibody.

Mouse lymphoid cells treated with multivalent hybrid antibody consisting of rabbit IgG anti-T antibody and rabbit IgG anti-chicken red blood cell antibody formed rosettes and killed specifically chicken red blood cells. The attachment and killing of chicken red blood cells is due to multivalent hybrid antibody which is able to link with one antibody specificity (anti-T) the T-lymphocytes and with the other one the chicken red blood cell surface antigen(s). Some indirect results seem to suggest natural killer cells as a candidate for the multivalent hybrid antibody-induced cytolytic effects. Multivalent hybrid antibody can be a useful alternative to mitogen for assaying specifically the cytotoxic potential of various cell populations pre-existing in normal organisms and requiring only a link to the target cells to reveal their killing ability.

Mutual inhibition of the binding of Clq and protein A to rabbit IgG immune complexes.

A complex of rabbit IgG antibody with horseradish peroxidase covalently linked to Sepharose 4B was used as an insoluble immune complex for studying the binding of complement factor C1q protein A from Staphylococcus aureus, and its IgG-binding fragments AB and B, to rabbit IgG. It was shown that protein A (mol. wt approx. 42,000) and fragments AB and B (mol. wts approx. 14,000 and 7000, respectively) inhibited the binding of C1q to insoluble immune complex at 4 degrees C. However, at 37 degrees C fragment B did not inhibit this binding. On the other hand, C1q, when bound to an insoluble immune complex, almost completely blocked the binding of protein A and fragment B at both temps. The higher affinity of C1q for its CH2-binding site than of fragment B for its CH2-binding site may explain the displacement of the latter from the CH2 domain. The mutual inhibition of the binding of C1q and protein A (and its smaller fragments) indicates that the binding sites for C1q and protein A are closely located in the CH2 domain.

Protein A vectorized toxins--II. Preparation and "in vitro" cytotoxic effect of protein A-ricin A chain conjugate on antibody coated human tumour cells.

Protein A of Staphylococcus aureus was covalently bound to reduced ricin A chain toxin by N-succinimidyl 3-(2-pyridyldithio)propionate. The conjugate consisting mainly of one molecule of protein A bound to two molecules of A chains (Mr 107,000) was purified by tandem affinity chromatography on ConA-Sepharose 4B and IgG-Sepharose 4B. The purified protein A-A chain conjugate was able to bind and kill human lymphoma cells coated either with monoclonal mouse IgG2a anti-kappa antibody or with polyclonal rabbit anti-kappa antibody. The cytotoxic activity of protein A-A chain conjugate in conjunction with either mouse or rabbit anti-kappa antibodies was 10 times higher than that of rabbit IgG anti-mouse IgG coupled with A chain on Daudi cells coated with mouse anti-kappa antibody and 100 times higher than that of rabbit anti-kappa antibody coupled with A chain on non-coated Daudi cells. The cytotoxic effect of protein A-A chain conjugate on antibody-coated Daudi cells (9 x 10(-12) M) was comparable with that of ricin toxin on non-coated Daudi cells (2 x 10(-12) M). The results recommend the use of protein A-ricin A chain toxin conjugate as a unique specific toxin for the "in vitro" killing of antibody-coated target cells.

Evidence that the hinge region plays a role in maintaining serum levels of the murine IgG1 molecule.

The site of the murine IgG1 molecule that regulates catabolism has recently been shown to encompass amino acids that are located at the CH2-CH3 domain interface. The CH2 and CH3 domains are connected to each other by a relatively flexible "mini-hinge" region, and flexibility in this region could clearly affect the orientation of the domains with respect to each other. The internal movement of the CH2 domain depends on the absence/presence of the hinge disulphide. The increased mobility of the CH2 domain relative to the CH3 domain in a hinge less IgG or Fc fragment may result in a conformational change at the CH2-CH3 domain interface and alter the accessibility of the residues that are involved in catabolism control. To investigate this possibility, four Fc fragments which differ in the presence/absence of hinge disulphides and hinge sequences have been analysed in both in vivo pharmacokinetic studies and in vitro by limited proteolysis with pepsin. The data show that the presence of hinge disulphide(s) in the Fc fragment results in a longer intravascular half life but a higher susceptibility to pepsin attack. This, taken together with the knowledge that pepsin cleaves close to the CH2-CH3 domain interface, suggests that the longer half life of disulphide linked Fc fragments relative to unlinked fragments may be due to conformational differences in this region of the IgG molecule, and these conformational changes may affect the accessibility of the catabolic site for binding to putative protective Fc receptors.

Protein A vectorized toxins--I. Preparation and properties of protein A-ricin toxin conjugates.

Protein A of Staphylococcus aureus (mol. wt 43,000) was covalently bound to ricin toxin (mol. wt 60,000) by N-succinimidyl 3-(2-pyridyldithio) propionate. The conjugate consisting of one molecule of protein A bound to one molecule of ricin toxin (mol. wt 100,000) was purified by successive affinity chromatographies on IgG-Sepharose 4B and ConA-Sepharose 4B. The purified protein A-ricin toxin conjugate was able to bind and kill IgG antibody coated leukemia EL4 cells leaving unaffected EL4 cells not coated with antibody. The cytotoxic efficacy of the conjugate was comparable to that of nonconjugated ricin toxin. The results recommend the use of protein A-ricin toxin conjugate as a "universal" specific toxin for the "in vitro" killing of various antibody-coated target cells.

The stoichiometry and affinity of the interaction of murine Fc fragments with the MHC class I-related receptor, FcRn.

The binding of recombinant wild type and mutant Fc-hinge fragments to soluble, FcRn expressed in insect cells has been analysed. The mutant Fc-hinge fragments are derived from murine IgG1 with mutation of residues located at the CH2-CH3 domain interface (Ile253, His31O, Gln311, His433 and Asn434; EU numbering). These mutant Fc-hinge fragments have previously been shown to be deficient in neonatal transcytosis in suckling mice and also have abnormally short serum half lives. The mutated residues are highly conserved in human and rodent gammaglobulins (IgGs) and are also involved in binding to staphylococcal protein A. This study demonstrates that the Fc mutants have lower binding affinities for recombinant FcRn and mutations in the CH2 domain have a greater effect than those in the CH3 domain. There is an excellent correlation between affinity and transcytosis or the control of catabolism, and this provides further evidence in support of the close overlap of the sites of IgG/Fc involved in these processes. The stoichiometry of the FcRn:Fc interaction has also been investigated and has been found to be 1:1, indicating that binding of FcRn to one CH2-CH3 domain interface site precludes an FcRn:Fc interaction at the second site.

Modulation of IgG effector functions by a monovalent fragment of staphylococcal protein A.

The monovalent V-1 fragment of protein A (fSpA) with a mol. wt of 13,000 obtained from an u.v. mutant of Staphylococcus aureus Cowan I strain was proved to be able to modulate significantly some of the effector functions of IgG, such as complement fixation, catabolism, attachment to Fc receptors and antibody-dependent cell-mediated cytotoxicity. Moreover fSpA-like protein A obtained from the A676 strain is mitogenic and enhances NK activity of human peripheral lymphocytes. The efficiency of fSpA was found to be lower than that of protein A with regard to its ability to inhibit complement fixation, EA rosette formation and antibody-dependent cell-mediated cytotoxicity. Both protein A and fSpA had the same efficiency in activation of the complement system after reaction with human or guinea pig IgG, and in increasing the IgG catabolism. Unlike fSpA the monovalent B fragment of protein A (with mol. wt of 7000) was not able to inhibit EA rosette formation and antibody-dependent cell-mediated cytotoxicity. The results recommend fSpA, substituting for protein A, as a molecular probe for the investigation of IgG antibody and lymphocyte effector functions.

Immunoconjugates containing ricin A chain and either human anti-gp41 or CD4 kill H9 cells infected with different isolates of HIV, but do not inhibit normal T or B cell function.

We have previously reported that immunoconjugates composed of deglycosylated ricin A chain coupled to either recombinant (r) CD4 or two different monoclonal human anti-gp41 antibodies (rCD4-dgA and anti-gp41-dgA, respectively) are specifically toxic to HIV-infected lines of human T cells (H9) and monocytes (U937). In order to further evaluate these immunoconjugates as potential therapeutic reagents for killing HIV-infected cells, H9 cells infected with five different isolates of HIV were used as target cells in vitro. All three HIV-specific immunoconjugates were toxic to H9 cells infected with each HIV isolate, but were virtually nontoxic to uninfected cells. Chloroquine markedly potentiated the specific toxicity of all three conjugates, particularly the anti-gp41-dgAs. None of the conjugates affected the ability of normal peripheral blood B cells to respond to mitogen or the ability of normal T cells to respond to alloantigens.

A probabilistic approach to ageing and elimination of senescent plasma protein.

A probabilistic mechanism of plasma protein ageing and elimination is suggested, based on the following assumptions: (1) ageing of plasma protein molecules is the result of their interaction with some microenvironmental factors, acting as disturbing factors; (2) a protein species is characterized by a definite spectrum of conformational substates, some of which, "altered" substates, are specifically recognized by a selective catabolic system and then compulsorily eliminated; (3) the disturbing (ageing) factors act by increasing the probability of reaching an "altered" substate. Based on these assumptions a mathematical model, giving the expression of the normalized catabolic rate as a function of the frequency and the effectiveness of disturbing impacts, is set up and some possible correlations with physicochemical data are discussed.

An immunological hypothesis for plasma protein catabolism.

A selective mechanism of plasma protein catabolism is suggested, based on three main assumptions: (1) plasma proteins are submitted to molecular ageing thus achieving "modified" forms after a given time period; (2) the system recognizing the "modified" molecules consists of preformed physiological autoantibodies; (3) the elimination of the immune complexes formed between "modified" proteins and the corresponding autoantibodies takes place via binding to Fc receptor bearing cells.