High-Throughput Cysteine Scanning To Identify Stable Antibody Conjugation Sites for Maleimide- and Disulfide-Based Linkers.

THIOMAB antibody technology utilizes cysteine residues engineered onto an antibody to allow for site-specific conjugation. The technology has enabled the exploration of different attachment sites on the antibody in combination with small molecules, peptides, or proteins to yield antibody conjugates with unique properties. As reported previously ( Shen , B. Q. , et al. ( 2012 ) Nat. Biotechnol. 30 , 184 - 189 ; Pillow , T. H. , et al. ( 2017 ) Chem. Sci. 8 , 366 - 370 ), the specific location of the site of conjugation on an antibody can impact the stability of the linkage to the engineered cysteine for both thio-succinimide and disulfide bonds. High stability of the linkage is usually desired to maximize the delivery of the cargo to the intended target. In the current study, cysteines were individually substituted into every position of the anti-HER2 antibody (trastuzumab), and the stabilities of drug conjugations at those sites were evaluated. We screened a total of 648 THIOMAB antibody-drug conjugates, each generated from a trastuzamab prepared by sequentially mutating non-cysteine amino acids in the light and heavy chains to cysteine. Each THIOMAB antibody variant was conjugated to either maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E (MC-vc-PAB-MMAE) or pyridyl disulfide monomethyl auristatin E (PDS-MMAE) using a high-throughput, on-bead conjugation and purification method. Greater than 50% of the THIOMAB antibody variants were successfully conjugated to both MMAE derivatives with a drug to antibody ratio (DAR) of >0.5 and <50% aggregation. The relative in vitro plasma stabilities for approximately 750 conjugates were assessed using enzyme-linked immunosorbent assays, and stable sites were confirmed with affinity-capture LC/MS-based detection methods. Highly stable conjugation sites for the two types of MMAE derivatives were identified on both the heavy and light chains. Although the stabilities of maleimide conjugates were shown to be greater than those of the disulfide conjugates, many sites were identified that were stable for both. Furthermore, in vitro stabilities of selected stable sites translated across different cytotoxic payloads and different target antibodies as well as to in vivo stability.

An Anti-GDNF Family Receptor Alpha 1 (GFRA1) Antibody-Drug Conjugate for the Treatment of Hormone Receptor-Positive Breast Cancer.

Luminal A (hormone receptor-positive) breast cancer constitutes 70% of total breast cancer patients. In an attempt to develop a targeted therapeutic for this cancer indication, we have identified and characterized Glial cell line-Derived Neurotrophic Factor (GDNF) Family Receptor Alpha 1 (GFRA1) antibody-drug conjugates (ADC) using a cleavable valine-citrulline-MMAE (vcMMAE) linker-payload. RNAseq and IHC analysis confirmed the abundant expression of GFRA1 in luminal A breast cancer tissues, whereas minimal or no expression was observed in most normal tissues. Anti-GFRA-vcMMAE ADC internalized to the lysosomes and exhibited target-dependent killing of GFRA1-expressing cells both in vitro and in vivo The ADCs using humanized anti-GFRA1 antibodies displayed robust therapeutic activity in clinically relevant cell line-derived (MCF7 and KPL-1) tumor xenograft models. The lead anti-GFRA1 ADC cross-reacts with rodent and cynomolgus monkey GFRA1 antigen and showed optimal pharmacokinetic properties in both species. These properties subsequently enabled a target-dependent toxicity study in rats. Anti-GFRA1 ADC is well tolerated in rats, as seen with other vcMMAE linker-payload based ADCs. Overall, these data suggest that anti-GFRA1-vcMMAE ADC may provide a targeted therapeutic opportunity for luminal A breast cancer patients. Mol Cancer Ther; 17(3); 638-49. ©2017 AACR.

Discovery of Peptidomimetic Antibody-Drug Conjugate Linkers with Enhanced Protease Specificity.

Antibody-drug conjugates (ADCs) have become an important therapeutic modality for oncology, with three approved by the FDA and over 60 others in clinical trials. Despite the progress, improvements in ADC therapeutic index are desired. Peptide-based ADC linkers that are cleaved by lysosomal proteases have shown sufficient stability in serum and effective payload-release in targeted cells. If the linker can be preferentially hydrolyzed by tumor-specific proteases, safety margin may improve. However, the use of peptide-based linkers limits our ability to modulate protease specificity. Here we report the structure-guided discovery of novel, nonpeptidic ADC linkers. We show that a cyclobutane-1,1-dicarboxamide-containing linker is hydrolyzed predominantly by cathepsin B while the valine-citrulline dipeptide linker is not. ADCs bearing the nonpeptidic linker are as efficacious and stable in vivo as those with the dipeptide linker. Our results strongly support the application of the peptidomimetic linker and present new opportunities for improving the selectivity of ADCs.

Pyrrolobenzodiazepine Dimer Antibody-Drug Conjugates: Synthesis and Evaluation of Noncleavable Drug-Linkers.

Three rationally designed pyrrolobenzodiazepine (PBD) drug-linkers have been synthesized via intermediate 19 for use in antibody-drug conjugates (ADCs). They lack a cleavable trigger in the linker and consist of a maleimide for cysteine antibody conjugation, a hydrophilic spacer, and either an alkyne (6), triazole (7), or piperazine (8) link to the PBD. In vitro IC50 values were 11-48 ng/mL in HER2 3+ SK-BR-3 and KPL-4 (7 inactive) for the anti-HER2 ADCs (HER2 0 MCF7, all inactive) and 0.10-1.73 µg/mL (7 inactive) in CD22 3+ BJAB and WSU-DLCL2 for anti-CD22 ADCs (CD22 0 Jurkat, all inactive at low doses). In vivo antitumor efficacy for the anti-HER2 ADCs in Founder 5 was observed with tumor stasis at 0.5-1 mg/kg, 1 mg/kg, and 3-6 mg/kg for 6, 8, and 7, respectively. Tumor stasis at 2 mg/kg was observed for anti-CD22 6 in WSU-DLCL2. In summary, noncleavable PBD-ADCs exhibit potent activity, particularly in HER2 models.

Novel antibody-antibiotic conjugate eliminates intracellular S. aureus.

Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. Here we confirm that intracellular reservoirs of S. aureus in mice comprise a virulent subset of bacteria that can establish infection even in the presence of vancomycin, and we introduce a novel therapeutic that effectively kills intracellular S. aureus. This antibody-antibiotic conjugate consists of an anti-S. aureus antibody conjugated to a highly efficacious antibiotic that is activated only after it is released in the proteolytic environment of the phagolysosome. The antibody-antibiotic conjugate is superior to vancomycin for treatment of bacteraemia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections.

A Novel Anti-CD22 Anthracycline-Based Antibody-Drug Conjugate (ADC) That Overcomes Resistance to Auristatin-Based ADCs.

PURPOSE: We are interested in identifying mechanisms of resistance to the current generation of antibody-drug conjugates (ADC) and developing ADCs that can overcome this resistance. EXPERIMENTAL DESIGN: Pinatuzumab vedotin (anti-CD22-vc-MMAE) and polatuzumab vedotin (anti-CD79b-vc-MMAE) are ADCs that contain the microtubule inhibitor monomethyl auristatin E (MMAE) attached to the antibody by the protease-cleavable linker maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-vc-PAB). Early clinical trial data suggest that these ADCs have promising efficacy for the treatment of non-Hodgkin lymphoma (NHL); however, some patients do not respond or become resistant to the ADCs. Anthracyclines are very effective in NHL, but ADCs containing the anthracycline doxorubicin were not clinically efficacious probably due to the low drug potency and inadequate linker technology. The anthracycline analogue PNU-159682 is thousands of times more cytotoxic than doxorubicin, so we used it to develop a new class of ADCs. We used the same MC-vc-PAB linker and antibody in pinatuzumab vedotin but replaced the MMAE with a derivative of PNU-159682 to make anti-CD22-NMS249 and tested it for in vivo efficacy in xenograft tumors resistant to MMAE-based ADCs. RESULTS: We derived cell lines from in vivo xenograft tumors that were made resistant to anti-CD22-vc-MMAE and anti-CD79b-vc-MMAE. We identified P-gp (ABCB1/MDR1) as the major driver of resistance to the vc-MMAE-based conjugates. Anti-CD22-NMS249 was at least as effective as anti-CD22-vc-MMAE in xenograft models of the parental cell lines and maintained its efficacy in the resistant cell lines. CONCLUSIONS: These studies provide proof of concept for an anthracycline-based ADC that could be used to treat B-cell malignancies that are resistant to vc-MMAE conjugates.

An anti-B7-H4 antibody-drug conjugate for the treatment of breast cancer.

B7-H4 has been implicated in cancers of the female reproductive system and investigated for its possible use as a biomarker for cancer, but there are no preclinical studies to demonstrate that B7-H4 is a molecular target for therapeutic intervention of cancer. We provide evidence that the prevalence and expression levels of B7-H4 are high in different subtypes of breast cancer and that only a few normal tissues express B7-H4 on the cell membrane. These profiles of low normal expression and upregulation in cancer provide an opportunity for the use of antibody-drug conjugates (ADCs), cytotoxic drugs chemically linked to antibodies, for the treatment of B7-H4 positive cancers. We have developed an ADC specific to B7-H4 that uses a linker drug consisting of a potent antimitotic, monomethyl auristatin E (MMAE), linked to engineered cysteines (THIOMAB) via a protease labile linker. We will refer to ADCs that use the THIOMAB format as TDCs to help distinguish the format from standard MC-vc-MMAE ADCs that are conjugated to the interchain disulfide bonds. Anti-B7-H4 (h1D11)-MC-vc-PAB-MMAE (h1D11 TDC) produced durable tumor regression in cell line and patient-derived xenograft models of triple-negative breast cancer. It also binds rat B7-H4 with similar affinity to human and allowed us to test for target dependent toxicity in rats. We found that our anti-B7-H4 TDC has toxicity findings similar to untargeted TDC. Our results validate B7-H4 as an ADC target for breast cancer and support the possible use of this TDC in the treatment of B7-H4(+) breast cancer.

The cryptophycins as potent payloads for antibody drug conjugates.

The cryptophycins are a potent class of cytotoxic agents that were evaluated as antibody drug conjugate (ADC) payloads. Free cryptophycin analog 1 displayed cell activity an order of magnitude more potent than approved ADC payloads MMAE and DM1. This potency increase was also reflected in the activity of the cryptophycin ADCs, attached via a either cleavable or non-cleavable linker.

Mechanism-Based Pharmacokinetic/Pharmacodynamic Model for THIOMAB™ Drug Conjugates.

PURPOSE: THIOMAB™ drug conjugates (TDCs) with engineered cysteine residues allow site-specific drug conjugation and defined Drug-to-Antibody Ratios (DAR). In order to help elucidate the impact of drug-loading, conjugation site, and subsequent deconjugation on pharmacokinetics and efficacy, we have developed an integrated mathematical model to mechanistically characterize pharmacokinetic behavior and preclinical efficacy of MMAE conjugated TDCs with different DARs. General applicability of the model structure was evaluated with two different TDCs. METHOD: Pharmacokinetics studies were conducted for unconjugated antibody and purified TDCs with DAR-1, 2 and 4 for trastuzumab TDC and Anti-STEAP1 TDC in mice. Total antibody concentrations and individual DAR fractions were measured. Efficacy studies were performed in tumor-bearing mice. RESULTS: An integrated model consisting of distinct DAR species (DAR0-4), each described by a two-compartment model was able to capture the experimental data well. Time series measurements of each Individual DAR species allowed for the incorporation of site-specific drug loss through deconjugation and the results suggest a higher deconjugation rate from heavy chain site HC-A114C than the light chain site LC-V205C. Total antibody concentrations showed multi-exponential decline, with a higher clearance associated with higher DAR species. The experimentally observed effects of TDC on tumor growth kinetics were successfully described by linking pharmacokinetic profiles to DAR-dependent killing of tumor cells. CONCLUSION: Results from the integrated model evaluated with two different TDCs highlight the impact of DAR and site of conjugation on pharmacokinetics and efficacy. The model can be used to guide future drug optimization and in-vivo studies.

Site-specific trastuzumab maytansinoid antibody-drug conjugates with improved therapeutic activity through linker and antibody engineering.

Antibody-drug conjugates (ADCs) have a significant impact toward the treatment of cancer, as evidenced by the clinical activity of the recently approved ADCs, brentuximab vedotin for Hodgkin lymphoma and ado-trastuzumab emtansine (trastuzumab-MCC-DM1) for metastatic HER2+ breast cancer. DM1 is an analog of the natural product maytansine, a microtubule inhibitor that by itself has limited clinical activity and high systemic toxicity. However, by conjugation of DM1 to trastuzumab, the safety was improved and clinical activity was demonstrated. Here, we report that through chemical modification of the linker-drug and antibody engineering, the therapeutic activity of trastuzumab maytansinoid ADCs can be further improved. These improvements include eliminating DM1 release in the plasma and increasing the drug load by engineering four cysteine residues into the antibody. The chemical synthesis of highly stable linker-drugs and the modification of cysteine residues of engineered site-specific antibodies resulted in a homogeneous ADC with increased therapeutic activity compared to the clinically approved ADC, trastuzumab-MCC-DM1.

Site-specific antibody drug conjugates for cancer therapy.

Antibody therapeutics have revolutionized the treatment of cancer over the past two decades. Antibodies that specifically bind tumor surface antigens can be effective therapeutics; however, many unmodified antibodies lack therapeutic activity. These antibodies can instead be applied successfully as guided missiles to deliver potent cytotoxic drugs in the form of antibody drug conjugates (ADCs). The success of ADCs is dependent on four factors--target antigen, antibody, linker, and payload. The field has made great progress in these areas, marked by the recent approval by the US Food and Drug Administration of two ADCs, brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (Kadcyla). However, the therapeutic window for many ADCs that are currently in pre-clinical or clinical development remains narrow and further improvements may be required to enhance the therapeutic potential of these ADCs. Production of ADCs is an area where improvement is needed because current methods yield heterogeneous mixtures that may include 0-8 drug species per antibody molecule. Site-specific conjugation has been recently shown to eliminate heterogeneity, improve conjugate stability, and increase the therapeutic window. Here, we review and describe various site-specific conjugation strategies that are currently used for the production of ADCs, including use of engineered cysteine residues, unnatural amino acids, and enzymatic conjugation through glycotransferases and transglutaminases. In addition, we also summarize differences among these methods and highlight critical considerations when building next-generation ADC therapeutics.

Engineering THIOMABs for site-specific conjugation of thiol-reactive linkers.

Antibody conjugates are used in many therapeutic and research applications and are generated by chemically linking a cysteine or lysine residue to potent chemotherapeutic drugs or other functional groups through a flexible linker. Recently, we have engineered THIOMABs (antibodies with engineered reactive cysteine residues) for site-specific conjugation and showed that these antibody conjugates display homogeneous labeling with optimal in vitro and in vivo characteristics. Here, we describe protocols for engineering, selection, and site-specific conjugation of THIOMABs with thiol-reactive linkers.

Total antibody quantification for MMAE-conjugated antibody-drug conjugates: impact of assay format and reagents.

Antibody-drug conjugates (ADCs) are target-specific anticancer agents consisting of cytotoxic drugs covalently linked to a monoclonal antibody. The number of ADCs in the clinic is growing, and therefore thorough characterization of the quantitative assays used to measure ADC concentrations in support of pharmacokinetic, efficacy, and safety studies is of increasing importance. Cytotoxic drugs such as the tubulin polymerization inhibiting auristatin, monomethyl auristatin E, have been conjugated to antibodies via cleavable linkers (MC-vc-PAB) through internal cysteines. This results in a heterogeneous mixture of antibody species with drug-to-antibody ratios (DAR) ranging from 0 to 8. In order to characterize the assays used to quantitate total MC-vc-PAB-MMAE ADCs (conjugated and unconjugated antibody), we used purified fractions with defined DARs from 6 therapeutic antibodies to evaluate different assay formats and reagents. Our investigations revealed that for quantitation of total antibody, including all unconjugated and conjugated antibody species, sandwich ELISA formats did not always allow for recovery of all purified DAR fractions (DAR 0-8) to within ±20% of the expected values at the reagent concentrations tested. In evaluating alternative approaches, we found that the recovery of DAR fractions with semihomogeneous assay (SHA) formats, in which sample, capture, and detection reagents are preincubated in solution, were less affected by the antibody's MMAE drug load as compared to traditional stepwise sandwich ELISAs. Thus, choosing the optimal assay format and reagents for total antibody assays is valuable for developing accurate quantitative assays.

The melanosomal protein PMEL17 as a target for antibody drug conjugate therapy in melanoma.

Melanocytes uniquely express specialized genes required for pigment formation, some of which are maintained following their transformation to melanoma. Here we exploit this property to selectively target melanoma with an antibody drug conjugate (ADC) specific to PMEL17, the product of the SILV pigment-forming gene. We describe new PMEL17 antibodies that detect the endogenous protein. These antibodies help define the secretory fate of PMEL17 and demonstrate its utility as an ADC target. Although newly synthesized PMEL17 is ultimately routed to the melanosome, we find substantial amounts accessible to our antibodies at the cell surface that undergo internalization and routing to a LAMP1-enriched, lysosome-related organelle. Accordingly, an ADC reactive with PMEL17 exhibits target-dependent tumor cell killing in vitro and in vivo.

Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates.

The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.

Characterization of intact antibody-drug conjugates from plasma/serum in vivo by affinity capture capillary liquid chromatography-mass spectrometry.

Antibody-drug conjugates (ADCs) are designed to facilitate the targeted delivery of cytotoxic drugs to improve their tumor fighting effects and minimize systemic toxicity. However, efficacy and safety can potentially be compromised due to the release of conjugated drugs from the ADC with time while in circulation, resulting in changes in the drug-to-antibody ratio (DAR). Current understanding of this process is limited because existing methods such as immunoassays fail to distinguish ADCs with different DARs. Here we demonstrate a novel method with bead-based affinity capture and capillary liquid chromatography-mass spectrometry to allow direct measurement of drug release by quantifying DAR distributions of the ADC in plasma/serum. This method successfully identified individual intact conjugated antibody species produced due to drug loss from ADCs (e.g., an engineered site-specific anti-MUC16 THIOMAB-drug conjugate) and measured the corresponding DAR distributions in vitro and in vivo. Information obtained can provide insights into the mechanisms involved in drug loss and help to optimize ADC therapeutics. Other potential applications of the method may include characterization of posttranslational modifications, protein adducts, and immunogenicity.

Engineered thio-trastuzumab-DM1 conjugate with an improved therapeutic index to target human epidermal growth factor receptor 2-positive breast cancer.

PURPOSE: Antibody drug conjugates (ADCs) combine the ideal properties of both antibodies and cytotoxic drugs by targeting potent drugs to the antigen-expressing tumor cells, thereby enhancing their antitumor activity. Successful ADC development for a given target antigen depends on optimization of antibody selection, linker stability, cytotoxic drug potency, and mode of linker-drug conjugation to the antibody. Here, we systematically examined the in vitro potency as well as in vivo preclinical efficacy and safety profiles of a heterogeneous preparation of conventional trastuzumab-mcc-DM1 (TMAb-mcc-DM1) ADC with that of a homogeneous engineered thio-trastuzumab-mpeo-DM1 (thioTMAb-mpeo-DM1) conjugate. EXPERIMENTAL DESIGN AND RESULTS: To generate thioTMAb-mpeo-DM1, one drug maytansinoid 1 (DM1) molecule was conjugated to an engineered cysteine residue at Ala114 (Kabat numbering) on each trastuzumab-heavy chain, resulting in two DM1 molecules per antibody. ThioTMAb-mpeo-DM1 retained similar in vitro anti-cell proliferation activity and human epidermal growth factor receptor 2 (HER2) binding properties to that of the conventional ADC. Furthermore, it showed improved efficacy over the conventional ADC at DM1-equivalent doses (µg/m(2)) and retained efficacy at equivalent antibody doses (mg/kg). An improved safety profile of >2-fold was observed in a short-term target-independent rat safety study. In cynomolgus monkey safety studies, thioTMAb-mpeo-DM1 was tolerated at higher antibody doses (up to 48 mg/kg or 6,000 µg DM1/m(2)) compared with the conventional ADC that had dose-limiting toxicity at 30 mg/kg (6,000 µg DM1/m(2)). CONCLUSIONS: The engineered thioTMAb-mpeo-DM1 with broadened therapeutic index represents a promising antibody drug conjugate for future clinical development of HER2-positive targeted breast cancer therapies.

In vivo effects of targeting CD79b with antibodies and antibody-drug conjugates.

Antibodies directed against B cells are in use for the treatment of non-Hodgkin's lymphoma and autoimmune disorders. The B-cell-restricted surface antigen CD79b, a signaling component of the B-cell receptor, has been shown as a promising antibody target in mouse efficacy models of systemic lupus erythematosus. Anti-CD79b antibody-drug conjugates (ADC), cytotoxic drugs linked through specialized chemical linkers to antibodies, are effective in mouse xenograft models of non-Hodgkin's lymphoma. We were interested in evaluating the systemic effects of anti-CD79b antibodies and ADCs in normal animals as a step toward the development of these molecules as therapeutics. As we were unable to identify any cell surface binding anti-human CD79b antibodies that were cross-reactive to other species, we developed an antibody to cynomolgus monkey (Macaca fascicularis) CD79b (anti-cyCD79b). The anti-cynomolgus antibody, anti-cyCD79b (10D10), and the maytansine (tubulin inhibitor)-conjugated ADC, anti-cyCD79b (10D10)-MCC-DM1, were administered to cynomolgus monkeys at approximately 30 mg/kg (6,000 microg DM1/m(2)) for two doses 3 weeks apart. Anti-cyCD79b and anti-cyCD79b-MCC-DM1 resulted in peripheral blood B-cell depletion of approximately 65% and approximately 94%, respectively. In addition, anti-cyCD79b-MCC-DM1 resulted in near-complete absence of splenic germinal centers, an observation supporting an effect on dividing B cells. Both molecules were well tolerated, with minimal findings for the antibody and findings for the ADC limited to the lymphoid and hematopoietic systems, liver, and peripheral nerves. These preclinical data suggest that targeting CD79b with antibodies or ADCs may provide safe and effective therapies for B-cell malignancies and autoimmune diseases.

A modular platform for the rapid site-specific radiolabeling of proteins with 18F exemplified by quantitative positron emission tomography of human epidermal growth factor receptor 2.

Receptor-specific proteins produced by genetic engineering are attractive as PET imaging agents, but labeling with conventional (18)F-based prosthetic groups is problematic due to long synthesis times, poor radiochemical yields, and low specific activities. Therefore, we developed a modular platform for the rapid preparation of water-soluble prosthetic groups capable of efficiently introducing (18)F into proteins. The utility of this platform is demonstrated by the thiol-specific prosthetic group, [(18)F]FPEGMA, which was used to produce site-specifically (18)F-labeled protein ((18)F-trastuzumab-ThioFab) in 82 min with a total radiochemical yield of 13 +/- 3% and a specific activity of 2.2 +/- 0.2 Ci/micromol. (18)F-trastuzumab-ThioFab retained the biological activity of native protein and was successfully validated in vivo with microPET imaging of Her2 expression in a xenograft tumor-bearing murine model modulated by the Hsp90 inhibitor, 17-(allylamino)-17-demethoxygeldanamycin.