Chemoimmunoconjugates

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For most cancers, traditional chemotherapy is still the standard of care. The selectivity of cytotoxic agents, however, relies on the premise that rapidly proliferating cells are more prone to the cytotoxic effect of these drugs. As a consequence, toxicities against normal tissues characterized by enhanced proliferation rates are regularly observed side effects which increase the potency of the drug. To reduce this risk, anticancer che-motherapeutics are often given at suboptimal doses. A strategy to achieve tumor selectivity and thereby circumvent this problem is conjugating cytotoxic agents to a tumor specific antibody; thereby, the mAb serves as a delivery vehicle for the targeted application of cytotoxic drugs. Such chemoimmunoconjugates indeed display selectivity towards the cells which express the respective antigens (Johnson et al. 1981). After binding, the conjugate is internalized via receptor-mediated endocytosis and the parent drug is released from the lysosome into the cell to restore its cytotoxic activity (Fig. 15.1).

Standard chemotherapeutic agents belonging to the antifolates, vinca alkaloids or anthracyclines have been conjugated to mAbs (Schrama et al. 2006). Although such immunoconj ugates based on standard anti-cancer drugs demonstrated efficiency in preclinical models (Deguchi et al. 1986), they were not efficient in the clinical situation. This was due to the fact that therapeutic levels of the cytotoxic agents within the cells were not

achieved since these drugs possessed only moderate cytotoxic potency. A high cytotoxic potency is important as both the amount of cytotoxic drug which can be conjugated to the antibody and the expression of antigens on the tumor cell is limited (Allen 2002). From these initial studies, however, crucial information was gathered concerning the influence of the targeting device on the pharmacokinetics of the conjugates as well as the different chemical strategies of coupling cytotoxic agents to the antibody. For example, peptide linkers, which are stable in serum but can be readily degraded in intracellular compartments by specific enzymes, were found to be superior to hydrazone linkers. In this respect, disulfide linkers are suitable for conjugation of drug and mAbs since these linkers are cleaved by disulfide exchange with an intracellular thiol such as glutathione; this is of particular advantage as the gluthatione concentration in tumor cells is generally higher than in normal cells (Jaracz et al. 2005). In addition, progress in DNA engineering, enabling the production of chimeric, humanized and human mAbs, solved the major problem of early antibody-drug conjugates, i.e., the rather high clearance rate of the immu-noconjugates due to the development of human anti-mouse antibody responses (HAMA) in the treated patients (Carter 2001).

Two avenues of improving the cytotoxic potency of antibody-drug conjugates have been pursued: (1)

Fig. 15.1. Internalization of antibody-drug conjugates. In order to regain their cytotoxic activity, the cytotoxic agent has to be cleaved from the chemoimmunoconjuga-te. Uptake of antibodies predominantly occurs via the clathrin mediated endocyto-sis pathway. After binding the respective antigen associated with coated pits, antibody-drug conjugates will readily be endocytosed, initiating their transit via several stages of transport and endosomal vesicles and finally ending up in a lyso-some. Subsequently, linkers and antibody will be cleaved, releasing the cytotoxic agent which - after exit from the lysosomal compartment -exerts its cytotoxic effect

Table 15.1. Immunoconjugates in clinical development2 (Schrama et al. 2006)

Name

Targeting device

Conjugate

Tumor

Phase

Immunoconjugates

Phase Ib

AVE9633 (huMy9 -

Humanized anti-CD33

DM4

AML

6-DM4)

mAb

BB-10901 (huN901-DM 1)

Humanized anti-CD56

DM1

Recurrent or refractory

Phase I and II

mAb

lung cancer or other

CD56+ solid tumors

CMC-544

Humanized anti-CD22

Calicheamicin

B-cell NHL

Phase I

mAb

Gemtuzumab ozogamicin

Humanized anti-CD33

Calicheamicin

Older patients with

Phase II and III

mAb

relapsed or untreated

AML

huC242-DM 4

Humanized anti-

DM4

CanAg+ solid tumors

Phase Ic

CanAg mAb

MLN2704

Humanized anti-PSMA

DM1

Prostate cancer

Phase I and II

mAb

SGN-15 with taxotere

Chimeric anti-Le(Y)

Doxorubicin

Prostate cancer

Phase II completed

mAb

ADEPT

A5CP + ZD2767P

Murine anti-CEA

Prodrug ZD2767P

Advanced CRC

Phase I

F(ab)2 fragment fused

to CPG2

MFECP1 + ZD2767P

Murine anti-CEA scFv

Prodrug ZD2767P

CEA expressing tumors

Phase I

fragment fused to

CPG2

Immunotoxins

BL22

Murine anti-CD22 dsFv

Truncated Pseudomo

Leukemia and lymphoma

Phase I and II

fragment

nas exotoxin A

Hum-195/rGel

Humanized anti-CD33

Recombinant gelonin

Advanced myeloid malig-

Phase I

antibody

nancies

LMB-2

Murine anti-CD25 scFv

Truncated Pseudomo-

Leukemia and lymphoma

Phase I and II

fragment

nas exotoxin A

LMB-9

Murine anti-Le(Y)

Truncated Pseudomo-

Advanced pancreatic,

Phase I completed

dsFv fragment

nas exotoxin A

esophageal, stomach can

cer or CRC

SS1(dsFv)-PE38

Murine anti-mesothe-

Truncated Pseudomo-

Mesothelin-expressing

Phase I

lin dsFv fragment

nas exotoxin

tumors like mesothelio-

ma, ovarian and pancre-

atic adenocarcinoma

Immunocytokines

EMD 273066

Humanized anti-

IL-2

Ovarian, prostate, CRC or

Phase I

EpCAM mAb

NSCL cancers

Bispecific double scFv

BiTE MT103

Rabbit anti-CD19 scFv

scFv fragment of a

B-cell tumors

Phase Id

fragment

murine anti-CD3 mAb

rM28

Murine anti-M-AP

scFV fragment of a

Metatstatic melanoma

Phase I and II;

scFv fragment

murine anti-CD28 mAb

not yet recruiting

a Information on ongoing trials was gathered from:

b http://utm-ext01a.mdacc.tmc.edu/dept/prot/clinicaltrialswp.nsf/Index/2004-0756 c http://www.idd.org/forms/PhaseI.pdf

d http://www.micromet.de or from http://www.clinicaltrials.gov [all others]. Status quo October 2005

AML acute myelogeneous leukemia, CPG2 carboxypeptidase 2, CRC colorectal cancer, ds disulfide-stabilized, M-AP melanoma-associated proteoglycan, NHL non-Hodgkin's lymphoma, NSCLC non-small cell lung cancer, PSMA prostate specific membrane antigen, ZD2767P bis-iodo phenol mustard increasing the number of molecules delivered per targeting moiety by means of carriers like liposomes and polymers, and/or (2) the use of highly cytotoxic compounds. Indeed, targeting extremely cytotoxic agents, such as calicheamicin, the maytansine derivative DM 1 or monomethyl auristatin E to the tumor, results in a pronounced anti-tumor activity in vivo. Nevertheless, to date only one immunoconjugate containing a cytotoxic drug has been approved by the FDA for the treatment of cancer. This immunoconjugate (Mylotarg) consists of a humanized anti-CD33 mAb (gemtuzu-mab) linked to the cytotoxic antibiotic N-acetyl-y-cali-cheamicin. Mylotarg is approved for the treatment of elderly patients relapsed from CD33-positive acute myeloid leukemia (AML) (Bross et al. 2001). In a multicenter phase II trial the combined response rate was around 30% with a median relapse-free survival of 7 months. A recent phase II trial demonstrated that Mylotarg can also be safely applied as first line therapy leading to objective responses in 27% of the patients (Nabhan et al. 2005). Notably, preliminary studies suggest that Mylotarg treatment may be potentiated by concomitant intensive chemotherapy. Consequently, a phase III trial is currently testing Mylotarg's impact on standard chemotherapy treatment in newly diagnosed AML patients (Tallman et al. 2005). Since Mylotarg demonstrated clinical efficiency in pediatric patients with advanced CD33-positive AML, further studies in combination with standard chemotherapy for this patient group are warranted.

Other cytotoxic drugs currently being investigated for use in drug-antibody conjugates include doxorubicin, DM 1, CC-1065, second generation taxane, mono-methylauristatin and geldanamycin E (Sanderson et al. 2005) (Table 15.1). For example, SGN-15, an antibody-doxorubicin conjugate in combination with docetaxel, is currently being tested in a phase II trial for patients with advanced non-small cell lung carcinoma (http://www.clinicaltrials.gov). The efficacy of therapeutic mAb maybe significantly improved by chemical conjugation with cytotoxic drugs; e.g., the Herceptin-geldanamycin conjugate demonstrated in a xenograft tumor model a much greater anti-tumor effect than the anti-HER2 mAb alone (Mandler et al. 2004).

The therapeutic efficiency of chemoimmunoconju-gates relies on their binding to and the subsequent internalization into the target cell. Thus, only tumor cells presenting the respective antigen are affected by this treatment. The antibody-directed enzyme prodrug therapy (ADEPT) is a strategy to overcome this limitation by expanding the anti-tumor effect towards cells not expressing the respective ligand. ADEPT is a targeted therapy where an enzyme is directed to the tumor -by a tumor specific antibody - which converts a weakly toxic prodrug into a very toxic agent (Bagshawe 1987). In contrast to antibody-drug conjugates, the antibody-enzyme conjugate has to remain on the cell surface after binding the respective antigen. In addition, the enzyme-antibody conjugate has to be cleared rapidly from the circulation to prevent toxicity. The latter is achieved by mannose glycosylation of the targeting moiety (Chester et al. 2004). Three classes of enzyme have been used for ADEPT (Senter and Springer 2001): (1) enzymes of non-mammalian origin with no mammalian homologs, (2) enzymes of non-mammalian origin with a mammalian homolog, and (3) enzymes of mammalian origin. Each class has both advantages and disadvantages. For example, prodrugs cleaved by class I enzymes are not cleavable by endogenous enzymes, which avoids toxicity against normal cells. However, due to their foreign nature, class I enzymes evoke a strong immune response. In contrast, class III enzymes are generally not immunogenic, but endogenous enzymes may cleave prodrugs designed for class III enzymes at inappropriate sites. Preclinical studies have proven the feasibility of ADEPT (Sharma et al. 2005); clinical data, however, are limited and largely restricted to phase I trials. An example is the prodrug ZD2767P: The activated form of ZD2767P proved to be highly cytotoxic, which gives a very short half-life (Francis et al. 2002). Despite the fact that the clinical efficiency for ADEPT remains to be established, the preclinical data demonstrating the capacity of this approach to reach high concentrations of cytotoxic drugs in the tumor, which will kill antigen negative tumor cells without severe systemic toxicity, warrants further testing.

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