Antibody Conjugates

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4.3.1. Genistein-Antibody Conjugate

Genistein, a tyrosine kinase inhibitor, has been linked to the anti-CD19 murine monoclonal antibody B43 (72). B43-genistein was given to one patient with CLL, but no antileukemic efficacy was seen. Dose-limiting toxicity was not reached.

4.3.2. Immunotoxins

4.3.2.1. Toxin Structure-Function. The immunotoxins constitute a novel class of therapeutics for CLL, with a distinct mechanism of action and toxicity. They target peptide ligands (monoclonal antibodies, antibody fragments, growth factors, or hormones) covalently attached

Fig. 1. Toxin three-dimensional structures. All molecules depicted are based on coordinates from Protein Data Bank (PDB) files. The PDB abbreviations are as follows: (A) 1ddt, diphtheria toxin; (B) 1dma, Pseudomonas exotoxin; (C) 2aa1, ricin; and (D) 1paf, pokeweed antiviral protein. SYBYL molecular modeling software was used to render toxins as shaded ribbons derived from cubic spline fits to the C-a backbone.

Fig. 1. Toxin three-dimensional structures. All molecules depicted are based on coordinates from Protein Data Bank (PDB) files. The PDB abbreviations are as follows: (A) 1ddt, diphtheria toxin; (B) 1dma, Pseudomonas exotoxin; (C) 2aa1, ricin; and (D) 1paf, pokeweed antiviral protein. SYBYL molecular modeling software was used to render toxins as shaded ribbons derived from cubic spline fits to the C-a backbone.

to polypeptide toxins. The antibody or other peptide ligand directs the molecule to the tumor cell surface, and the toxin moiety then enters the cell and induces apoptosis by inactivating protein synthesis. Extremely potent catalytic toxins that can kill cells with as few as one molecule per cell are found in plants, bacteria, and fungi. The atomic three-dimensional structures and the genes for a number of toxins used clinically and in the laboratory have been defined (Fig. 1). Many of the peptide toxins have three domains. These include a catalytic domain that enzymatically inactivates protein synthesis, a translocation domain that facilitates the transfer of the catalytic domain to the cytosol, where it can work, and a receptor-binding domain that directs the molecule to the cell surface, and, in some cases, triggers receptor-mediated endocytosis.

Diphtheria toxin (DT), produced by Corynebacterium diphtheriae, is a 58-kDa protein. DT is composed of an N-terminal adenosine diphosphate (ADP)-ribosylating catalytic domain, a furin-sensitive RVRR peptide (within a disulfide loop), a hydrophobic middle domain (responsible for translocation of the ADP-ribosylation domain to the cytosol), and a C-terminal cell-binding domain [capable of binding cell-associated heparin-binding epidermal growth factor (EGF)] (73). The N-terminal first 10 amino acid residues of DT may also be required for translocation of the catalytic domain to the cytosol (107). One or more pairs of amphipathic a-helices in the transmembrane middle domain of DT are involved in membrane insertion and channel formation.

Pseudomonas exotoxin (PE) is a product of Pseudomonas aeruginosa. It is a 68-kDa protein with an N-terminal domain (which binds the a2-macroglobulin receptor/low-density lipoprotein-like receptor protein), a furin-sensitive RQPRG sequence, a transmembrane domain with

DT RICIN/PE

DT RICIN/PE

Fig. 2. Mechanisms of cell intoxication by toxins. Toxins bind to cell surface receptors; the complex internalizes to endosomes; the toxin reaches a translocation-competent compartment [endosomes for diphtheria toxin (DT) and endoplasmic reticulum for Pseudomonas exotoxin (PE) and ricin); the catalytic domain of the toxin crosses the membrane to the cytosol; cytosolic toxin inactivates protein synthesis. DTA, diphtheria toxin A fragment; PEIII, Pseudomonas exotoxin A; and RTA, ricin toxin A.

Fig. 2. Mechanisms of cell intoxication by toxins. Toxins bind to cell surface receptors; the complex internalizes to endosomes; the toxin reaches a translocation-competent compartment [endosomes for diphtheria toxin (DT) and endoplasmic reticulum for Pseudomonas exotoxin (PE) and ricin); the catalytic domain of the toxin crosses the membrane to the cytosol; cytosolic toxin inactivates protein synthesis. DTA, diphtheria toxin A fragment; PEIII, Pseudomonas exotoxin A; and RTA, ricin toxin A.

amphipathic helices (that participate in membrane translocation), a catalytic domain [which ADP-ribosylates elongation factor-2 (EF2) similar to the DT catalytic fragment], a C-terminal REDLK sequence (with the REDL recognizable by the KDEL receptor), and a C-terminal lysine (sensitive to intracellular carboxy-peptidase) (74,108,109).

Ricin toxin from Ricinus communis plant seeds has two separate polypeptide chains linked by a disulfide bond. The B chain (RTB) is a 33-kDa glycoprotein lectin that binds three galactose-terminated oligosaccharides on cell surfaces (75). The A chain (RTA) is a 32-kDa glycoprotein possessing an active site cleft with RNA N-glycosidase activity acting on the critical stem-loop structure of rRNA involved in EF2 binding, and a C-terminal membrane insertion signal sequence (ILIPIIALMVY).

Pokeweed antiviral protein (PAP) and saporin (SAP) are type I ribosome-inactivating proteins (RIPs) isolated from the seeds of Phytolacca americana and Saponaria officinalis, respectively. The three-dimensional structures of these 28-kDa proteins closely resemble RTA, and the proteins have similar enzymatic activity (76).

These proteins intoxicate mammalian cells by a number of discrete steps mediated by different portions of the molecules (Fig. 2). The first step is cell surface binding. DT binds a membrane form of heparin-binding EGF (77). PE binds the a2-macroglobulin low density lipoprotein-like receptor protein (78). Ricin is a lectin with specificity for galactosyl pyranoside groups on cell surface glycoproteins and glycolipids (79). The plant hemitoxins or type I RIPs lack cell-binding domains. The second step is internalization into endosomes. The third step is transfer to a compartment for membrane translocation. In the case of DT, the acidic environment of the endosomes triggers alterations in the structure of the translocation domain of DT (80). In the case of PE and ricin, toxins pass to the trans-reticular Golgi and then travel by retrograde transport through the secretory pathways to the endoplasmic reticulum (81). The fourth step is membrane translocation. The enzymatic domain of DT passes through channels created by the transmembrane domain

(82). In the case of ricin and PE, the enzymatic domain may use pre-existing pores in the endoplasmic reticulum (the translocon) to reach the cytosol. The third and fourth steps taken by type I RIPs are unknown. The final step is inactivation of protein synthesis and apoptosis. Apoptosis facilitates but is not absolutely necessary for cell death (110).

We will briefly review the molecular steps in protein synthesis to define the toxin targets better. After the initiation step, an amino-acyl-tRNA residue (Met-tRNA) resides at the ribosomal A site. Later, during protein synthesis, there may be a peptide chain attached to this tRNA. The driving machine for peptide elongation is the transfer of this amino-acyl tRNA or peptidyl-tRNA from the A site to the ribosomal P site. This step is driven by guanosine triphosphate (GTP) and molecular mimicry. EF2 imitates a tRNA-EF1 complex. This complex has a higher affinity for the A site than the amino-acyl or peptidyl-tRNA alone. The peptide-tRNA is driven off the A site to the P site, facilitated by rotational movement between the subunits centered on the A site and replaced by the EF2 itself (83). DT and PE enzymatically ADP-ribosylate EF2 at the diphthamide residue located at D575 in domain IV of EF2 (84). The catalysis occurs at 100 or more EF2 molecules/min (from a total of about 105 EF2 molecules/cell). The chemical change prevents the EF2 domain IV probe, which mimics tRNA, from functioning normally and pushing the tRNA-peptide out of the A site. In addition to the EF2 probe, which displaces the peptidyl-tRNA, there is also the rRNA conformation change, which occurs spontaneously to drive elongation, but which is speeded by other EF2 domains (II-III) in which the GTPase activity modifies structure. Ricin and type I RIPs remove an adenine base (rRNA N-glycosidase activity) from the large rRNA at the S/R stem-loop (S = a-sarcin site and R = ricin site) (85). This stem-loop normally switches between two states, which changes overall rRNA and ribosome structure in the large subunit. Once this chemical change occurs (again at 100/min), EF2 can no longer bind properly, and elongation stops. Note that EF2 interacts with the ribosome at two sites, each of which is affected separately by peptide toxins. Once protein synthesis is inhibited, programmed cell death (apoptosis) occurs by unknown signaling pathways.

4.3.2.2. Immunotoxin Synthesis. Important steps for the construction of immunotoxins are to remove normal tissue-binding sites from the toxin and to identify adequately specific ligands. These ligands must also have the property of facilitating internalization of the ligand-receptor complex once bound to the cell surface. Finally, the ligand and toxin must be covalently linked together in such a way that both ligand and non-cell-binding toxin functions are preserved.

Normal tissue binding sites of DT, PE, and ricin can be genetically removed or mutated to reduce normal tissue binding markedly. For DT, this is accomplished by genetically deleting the C-terminal receptor binding domain (amino acids 389-535) to produce DAB389 or DT388 or by mutating critical amino acid residues in this domain such as CRM107 (S525F). PE is genetically modified by removing domain IA and IB sequences (amino acids 1-252 and 364-380), resulting in PE38, or replacing the four lysines of domain IA with glutamic acids (PEGlu4). The normal ricin tissue-binding domain is the B chain. This can be removed by genetic expression of the A chain alone and by mutating critical residues in the RTB lectin pockets. Ricin can also be manipulated chemically. The ricin can be extracted from the castor beans and purified on matrices; then the ricin is reduced and the RTB is discarded. The RTA can then be further purified from contaminating ricin. Alternatively, ricin can be derivatized to block its lectin sites with oligosaccharides containing a reactive dichlorothiazine group. Since the type I RIPs lack normal tissue-binding sites, no modifications are needed.

The identification of CLL-selective antigens and receptors for targeting is the most difficult step. Some ligands have been chosen for preclinical and clinical development of CLL

Table 3 CLL Immunotoxins

Conjugate

Ligand

Toxin

Linker

Properties (IC 50)

Ref.

RFB4(dsFv)-PE

Anti-CD22 dsFv

PE

Amide

160 pM on CLL cells; active on cells with 350 CD22 sites/cell

86

Anti-CD22-RTA

Anti-CD22 MAb

RTA

SPDP

10,000 pM on CLL cells

87

Anti-Tac(Fv)-PE38

Anti-CD25 sFv

PE

Amide

100 pM; active on cells with only CD25

88

DAB389lL-2/DT388IL-2

IL-2

DT

Amide

2 pM on HUT102 cells; 1500 pM on CLL cells

89,90

DAB486IL-2

IL-2

DT

Amide

100 pM on HUT102 cells; requires IL-2Ra, -b, -g; not tested on CLL

91

DAB389IL-7

IL-7

DT

Amide

1000 pM; requires IL-7Ra, -b, not tested on CLL cells

92

Anti-cCLLa-RTA

Anti-cCLLa MAb

RTA

SPDP

100 pM on CLL cells

93

Anti-CD19-bRicin

Anti-CD19 MAb

bRicin

SMCC

20 pM on Namalwa cells, not tested on CLL cells

94

Anti-CD19-genistein

Anti-CD19 MAb

Genistein

SANPAH

75 nM on B-ALL cell lines; not tested on CLL cells; requires CD19

95

OM124-rRTA

Anti-CD22 MAb

rRTA

SMPT

>10,000 pM on CLL cells; 20 pM with 10 nM monensin

96

OM124/saporin

Anti-CD22 MAb

Saporin

SMPT

70 pM on Daudi cells, not tested on CLL cells

97

T101-RTA

Anti-CD5

RTA

SPDP

>10,000 pM on CLL cells; 10 pM with 50 nM HSA-monensin

98

RTA, ricin toxin A chain; MAb, monoclonal antibody; bRicin, blocked ricin; PE, Pseudomonas exotoxin A; IL-2, interleukin-2; PE38, 38-kDa fragment of PE; DAB389, 389 N-terminal amino acid residues of diphtheria toxin; DT, diphtheria toxin; sFv, single-chain Fv fragment of MAb; dsFv, disulfide-stabilized sFv; SPDP, 3-(2-pyridyldithio)propionic acid V-hydroxysuccinimide ester; HSA, human serum albumin; SMCC, 4-(maleimidomethyl)cyclohexanecarboxylic acid V-hydroxysuccinimide ester; pM, picomolar; IL-2R, interleukin-2 receptor; SANPAH, V-succinimidyl-6(4'-azido-2'-nitrophenylamino)hexanoate; rRTA, recombinant RTA; SMPT, 4-succinimidyloxylcarbonyl-a-methyl-a-(2-pyridyldithio)-toluene; SPDP, V-succinimidyl 3-(2-pyrridyldithio) propronate.

RTA, ricin toxin A chain; MAb, monoclonal antibody; bRicin, blocked ricin; PE, Pseudomonas exotoxin A; IL-2, interleukin-2; PE38, 38-kDa fragment of PE; DAB389, 389 N-terminal amino acid residues of diphtheria toxin; DT, diphtheria toxin; sFv, single-chain Fv fragment of MAb; dsFv, disulfide-stabilized sFv; SPDP, 3-(2-pyridyldithio)propionic acid V-hydroxysuccinimide ester; HSA, human serum albumin; SMCC, 4-(maleimidomethyl)cyclohexanecarboxylic acid V-hydroxysuccinimide ester; pM, picomolar; IL-2R, interleukin-2 receptor; SANPAH, V-succinimidyl-6(4'-azido-2'-nitrophenylamino)hexanoate; rRTA, recombinant RTA; SMPT, 4-succinimidyloxylcarbonyl-a-methyl-a-(2-pyridyldithio)-toluene; SPDP, V-succinimidyl 3-(2-pyrridyldithio) propronate.

Table 4

Clinical Trials of Immunotoxins

Table 4

Clinical Trials of Immunotoxins

Agent

Criteria

Responses (PR + CR)/total

Toxicities

Ref.

T101-RTA

CD5+

0/9

Fever and nausea

98, 99

Anti-CD19-blocked ricin

CD19+

0/5

Transaminasemia, VLS, nausea, headaches

100, 101

DAB486IL-2

CD25+ in one

1/7

Transaminasemia, uremia, proteinuria,

102-104

of two trials

fever, chills, bronchospasm

DAB389IL-2

CD25+

2/2

None

Frankel, unpublished data

Anti-Tac(Fv)-PE38

CD25+

1/8

Transaminasemia, fever

105

Anti-CD22(dsFv)-PE38

CD22+

1/1

HUS, VLS, nausea, transaminasemia,

106

myalgias in other patients

VLS, vascular leak syndrome with hypoalbuminemia, edema, hypotension, weight gain; PR, partial response; CR, complete response; HUS, hemolytic-uremic syndrome.

VLS, vascular leak syndrome with hypoalbuminemia, edema, hypotension, weight gain; PR, partial response; CR, complete response; HUS, hemolytic-uremic syndrome.

immunotoxins, even though they are not truly tumor-specific. Instead, all these ligands bind antigens or receptors enriched on CLL cells and not significantly present on vital normal tissues.

Finally, conjugation of the ligand to toxin can be achieved by amide linkage using genetic engineering or by chemical crosslinking using bifunctional reagents such as the thiolating compounds [SPDP, SMPT (see Table 3 footnote), and MBS(m-maleidobenzoyl-N-hydroxy-succinimide ester)]. The critical tests are as follows: (1) the genetic or chemical modification must not change the functions of the ligand or toxin significantly, and (2) the conjugate must be efficiently internalized from the cell surface. A list of active immunotoxins targeted to CLL cells is shown in Table 3. Some of these have been developed and tested clinically.

4.3.2.3. Clinical Studies of Immunotoxin in CLL. There have been only a few limited pilot studies of immunotoxin therapy for CLL. This is surprising considering the excellent attributes of this disease as a target for immunotoxins. The patients will not mount an immune response against the foreign protein; the leukemic target is easily accessible to the drug in the blood, spleen, marrow, and lymph nodes. The studies are listed in Table 4 and detailed below. The immunotoxins tested to date include anti-CD5-RTA, DAB486IL2, DAB389IL2 (ONTAK), anti-CD19-blocked ricin, anti-CD25(Fv)-PE38 (LMB-2), and anti-CD22(dsFv)-PE38. In all the trials, only 35 CLL patients have received a variety of immunotoxins. Clinical efficacy was observed only with the fusion proteins containing fragments of the bacterial toxins—diphtheria toxin and Pseudomonas exotoxin. The responses were partial remissions and lasted for months. Activity to date has been modest with only rare partial remissions, in most cases. Reasons for the modest activity may be short treatment courses, low doses, dose-limiting vascular or hepatic injury, and, in some cases, poor patient selection with patients included who lacked high antigen density or high-affinity receptors. Nevertheless, more extensive phase II studies are warranted, with adjustment for these variables to define the true activity of these agents in this disease. The observation with rituximab that combination therapies with cytotoxic drugs markedly enhanced the overall and complete remission rate should serve as a guide for immunotoxin development. Like humanized monoclonal antibodies, immunotoxins have a distinct mechanism of action and toxicity profile. Once sufficient numbers of CLL patients are treated on phase I/II trials of immunotoxins, they should be tested in combination with cytotoxic drugs.

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