Jane S. Forsyth

Synthesis of site-specific antibody-drug conjugates using unnatural amino acids

Antibody-drug conjugates (ADCs) allow selective targeting of cytotoxic drugs to cancer cells presenting tumor-associated surface markers, thereby minimizing systemic toxicity. Traditionally, the drug is conjugated nonselectively to cysteine or lysine residues in the antibody. However, these strategies often lead to heterogeneous products, which make optimization of the biological, physical, and pharmacological properties of an ADC challenging. Here we demonstrate the use of genetically encoded unnatural amino acids with orthogonal chemical reactivity to synthesize homogeneous ADCs with precise control of conjugation site and stoichiometry. p-Acetylphenylalanine was site-specifically incorporated into an anti-Her2 antibody Fab fragment and full-length IgG in Escherichia coli and mammalian cells, respectively. The mutant protein was selectively and efficiently conjugated to an auristatin derivative through a stable oxime linkage. The resulting conjugates demonstrated excellent pharmacokinetics, potent in vitro cytotoxic activity against Her2+ cancer cells, and complete tumor regression in rodent xenograft treatment models. The synthesis and characterization of homogeneous ADCs with medicinal chemistry-like control over macromolecular structure should facilitate the optimization of ADCs for a host of therapeutic uses.

Platelet glycoprotein Ibα supports experimental lung metastasis

The platelet paradigm in hemostasis and thrombosis involves an initiation step that depends on platelet membrane receptors binding to ligands on a damaged or inflamed vascular surface. Once bound to the surface, platelets provide a unique microenvironment supporting the accumulation of more platelets and the elaboration of a fibrin-rich network produced by coagulation factors. The platelet-specific receptor glycoprotein (GP) Ib-IX, is critical in this process and initiates the formation of a platelet-rich thrombus by tethering the platelet to a thrombogenic surface. A role for platelets beyond the hemostasis/thrombosis paradigm is emerging with significant platelet contributions in both tumorigenesis and inflammation. We have established congenic (N10) mouse colonies (C57BL/6J) with dysfunctional GP Ib-IX receptors in our laboratory that allow us an opportunity to examine the relevance of platelet GP Ib-IX in syngeneic mouse models of experimental metastasis. Our results demonstrate platelet GP Ib-IX contributes to experimental metastasis because a functional absence of GP Ib-IX correlates with a 15-fold reduction in the number of lung metastatic foci using B16F10.1 melanoma cells. The results demonstrate that the extracellular domain of the α-subunit of GP Ib is the structurally relevant component of the GP Ib-IX complex contributing to metastasis. Our results support the hypothesis that platelet GP Ib-IX functions that support normal hemostasis or pathologic thrombosis also contribute to tumor malignancy.

Reduced surface expression and binding of fibronectin by thrombin-stimulated thrombasthenic platelets.

Thrombin stimulation results in increased surface expression of endogeneous fibronectin and binding of plasma fibronectin to human platelets. Platelets of patients with Glanzmanns thrombasthenia, a bleeding disorder, exhibit reduced thrombin-induced platelet aggregation, little or no clot retraction, and abnormal platelet spreading on glass surfaces. Thrombin stimulation of patient platelets from four thrombasthenic kindreds resulted in little fibronectin binding. Nevertheless, thrombin did induce serotonin secretion from these cells, indicating that stimulation was occurring. Thrombasthenic platelets did not inhibit thrombin-stimulated fibronectin binding to coincubated normal cells, suggesting that their defect was not due to the presence of a soluble inhibitor of fibronectin binding. Thrombin-stimulated afibrinogenemic platelets bound similar quantities of fibronectin to normal cells, indicating that the thrombasthenic deficit is not secondary to reduced fibrinogen content or binding. The thrombasthenic cells had an endogenous fibronectin content of 2.9 +/- 0.7 micrograms/10(9) platelets, whereas cells simultaneously prepared from five normal individuals contained 1.8 +/- 0.7 micrograms/10(9) platelets, a statistically insignificant difference. Nevertheless, thrombin stimulation did not increase expression of endogeneous fibronectin antigen on the surface of the thrombasthenic platelets as judged by immunofluorescence. These defects in platelet fibronectin binding and surface expression may account for some of the manifestations of Glanzmanns thrombasthenia.

Selective Formation of Covalent Protein Heterodimers with an Unnatural Amino Acid

We report a strategy for the generation of heterodimeric protein conjugates using an unnatural amino acid with orthogonal reactivity. This paper addresses the challenges of site-specificity and homogeneity with respect to the synthesis of bivalent proteins and antibody-drug conjugates. There are numerous antibody-drug conjugates in preclinical and clinical development, yet these are based either on nonspecific lysine coupling chemistry or on disulfide modification made difficult by the large number of cysteines in antibodies. Here, we describe a recombinant approach that can be used to rapidly generate a variety of constructs with defined conjugation sites. Moreover, this methodology results in homogeneous antibody conjugates whose biological, physical, and pharmacological properties can be quantitatively assessed and subsequently optimized. As proof of concept, we have generated anti-Her2 Fab-Saporin conjugates that demonstrate excellent potency in vitro.

A CXCR4-Targeted Site-Specific Antibody-Drug Conjugate

A chemically defined anti-CXCR4-auristatin antibody-drug conjugate (ADC) was synthesized that selectively eliminates tumor cells overexpressing the CXCR4 receptor. The unnatural amino acid p-acetylphenylalanine (pAcF) was site-specifically incorporated into an anti-CXCR4 immunoglobulin G (IgG) and conjugated to an auristatin through a stable, non-cleavable oxime linkage to afford a chemically homogeneous ADC. The full-length anti-CXCR4 ADC was selectively cytotoxic to CXCR4(+) cancer cells in vitro (half maximal effective concentration (EC50 )≈80-100 pM). Moreover, the anti-CXCR4 ADC eliminated pulmonary lesions from human osteosarcoma cells in a lung-seeding tumor model in mice. No significant overt toxicity was observed but there was a modest decrease in the bone-marrow-derived CXCR4(+) cell population. Because CXCR4 is highly expressed in a majority of metastatic cancers, a CXCR4-auristatin ADC may be useful for the treatment of a variety of metastatic malignancies.

Nicotinamide phosphoribosyltransferase can affect metastatic activity and cell adhesive functions by regulating integrins in breast cancer

NAD(+) metabolism is an essential regulator of cellular redox reactions, energy pathways, and a substrate provider for NAD(+) consuming enzymes. We recently demonstrated that enhancement of NAD(+)/NADH levels in breast cancer cells with impaired mitochondrial NADH dehydrogenase activity, through augmentation of complex I or by supplementing tumor cell nutrients with NAD(+) precursors, inhibits tumorigenicity and metastasis. To more fully understand how aberrantly low NAD(+) levels promote tumor cell dissemination, we here asked whether inhibition of NAD(+) salvage pathway activity by reduction in nicotinamide phosphoribosyltransferase (NAMPT) expression can impact metastasis and tumor cell adhesive functions. We show that knockdown of NAMPT, the enzyme catalyzing the rate-limiting step of the NAD(+) salvage pathway, enhances metastatic aggressiveness in human breast cancer cells and involves modulation of integrin expression and function. Reduction in NAMPT expression is associated with upregulation of select adhesion receptors, particularly αvβ3 and β1 integrins, and results in increased breast cancer cell attachment to extracellular matrix proteins, a key function in tumor cell dissemination. Interestingly, NAMPT downregulation prompts expression of integrin αvβ3 in a high affinity conformation, known to promote tumor cell adhesive interactions during hematogenous metastasis. NAMPT has been selected as a therapeutic target for cancer therapy based on the essential functions of this enzyme in NAD(+) metabolism, cellular redox, DNA repair and energy pathways. Notably, our results indicate that incomplete inhibition of NAMPT, which impedes NAD(+) metabolism but does not kill a tumor cell can alter its phenotype to be more aggressive and metastatic. This phenomenon could promote cancer recurrence, even if NAMPT inhibition initially reduces tumor growth.

Comparison of in vitro and in vivo approaches to studying brain colonization by breast cancer cells

Brain metastases occur in 20 to 40% of patients with metastatic breast cancer. The process is complex and depends on successful cancer cell evasion from the primary tumor, distribution and survival within the blood stream and cerebral microvasculature, penetration of the blood brain barrier and proliferation within the brain microenvironment. The initial steps of brain colonization are difficult to study in vivo. Therefore, in vitro assays have been developed to mimic this process. Most commonly, in vitro studies of brain colonization focus on tumor cell adhesion to brain endothelial cells and transendothelial migration. We previously investigated breast cancer brain colonization from the blood stream in vivo and defined the time and process of brain entry for five different cancer cell lines in a mouse model. We now investigated if in vitro approaches can reliably emulate the initial steps that determine successful brain colonization in vivo. To this end, we optimized an in vitro model of the vascular blood brain barrier and compared the brain invasion properties of the in vivo characterized cell models with their ability to interact with and penetrate the blood brain barrier model in vitro. Our results show that the in vitro findings correlate only poorly with the vivo results. The limitations of the in vitro approaches are discussed in light of the in vivo processes. We conclude that investigation of mechanisms supporting the earliest steps of breast cancer brain metastasis from the blood stream will depend on in vivo analyses.

Targeting activated integrin αvβ3 with patient-derived antibodies impacts late-stage multiorgan metastasis

Advanced metastatic disease is difficult to manage and specific therapeutic targets are rare. We showed earlier that metastatic breast cancer cells use the activated conformer of adhesion receptor integrin αvβ3 for dissemination. We now investigated if targeting this form of the receptor can impact advanced metastatic disease, and we analyzed the mechanisms involved. Treatment of advanced multi-organ metastasis in SCID mice with patient-derived scFv antibodies specific for activated integrin αvβ3 caused stagnation and regression of metastatic growth. The antibodies specifically localized to tumor lesions in vivo and inhibited αvβ3 ligand binding at nanomolar levels in vitro. At the cellular level, the scFs associated rapidly with high affinity αvβ3 and dissociated extremely slowly. Thus, the scFvs occupy the receptor on metastatic tumor cells for prolonged periods of time, allowing for inhibition of established cell interaction with natural αvβ3 ligands. Potential apoptosis inducing effects of the antibodies through interaction with caspase-3 were studied as potential additional mechanism of treatment response. However, in contrast to a previous concept, neither the RGD-containing ligand mimetic scFvs nor RGD peptides bound or activated caspase-3 at the cellular or molecular level. This indicates that the treatment effects seen in the animal model are primarily due to antibody interference with αvβ3 ligation. Inhibition of advanced metastatic disease by treatment with cancer patient derived single chain antibodies against the activated conformer of integrin αvβ3 identifies this form of the receptor as a suitable target for therapy.

Multiple catalytic aldolase antibodies suitable for chemical programming.

Chemical programming of nine murine antibodies with catalytic aldolase activity was examined using compounds, equipped with diketone or pro-vinyl ketone linkers that inhibit integrin adhesion receptor functions. The results showed that most Abs were programmed using the diketone compounds in a manner similar to previously reported catalytic antibody 38C2. On the other hand, only those antibodies, which catalyzed the retro aldol reaction of the pro-vinyl ketone linkers efficiently, were programmed. Conjugated to integrin targeting compounds, at least three new antibodies, including 84G3, 85H6, and 90G8, exhibited high specific binding to human tumor cells expressing integrin alpha(v)beta(3.).

Chemically Programmed Antibodies Targeting Multiple Alpha(v) Integrins and Their Effects on Tumor-Related Functions in vitro

Integrins αvβ3 and αvβ6 are highly expressed on tumor cells and/or by the tumor vasculature of many human cancers, and represent promising targets for anticancer therapy. Novel chemically programmed antibodies (cpAbs) targeting these integrins were prepared using the catalytic aldolase Antibody (Ab) programming strategy. The effects of the cpAbs on cellular functions related to tumor progression were examined in vitro using tumor cell lines and their cognate integrin ligands, fibronectin and osteopontin. The inhibitory functions of the conjugates and their specificity were examined based on interference with cell-cell and cell-ligand interactions related to tumor progression. Cell binding analyses of the anti-integrin cpAbs revealed high affinity for tumor cells that overexpressed αvβ3 and αvβ6 integrins, and weak interactions with αvβ1 and αvβ8 integrins, in vitro. Functional analyses demonstrated that the cpAbs strongly inhibited cell-cell interactions through osteopontin binding, and they had little or no immediate effects on cell viability and proliferation. On the basis of these characteristics, the cpAbs are likely to have a broad range of activities in vivo, as they can target and antagonize one or multiple αv integrins expressed on tumors and tumor vasculatures. Presumably, these conjugates may inhibit the establishment of metastastatic tumors in distant organs through interfering with cell adhesion more effectively than antibodies or compounds targeting one integrin only. These anti-integrin cpAbs may also provide useful reagents to study combined effect of multiple αv integrins on cellular functions in vitro, on pathologies, including tumor angiogenesis, fibrosis, and epithelial cancers, in vivo.