Novel immunoconjugates comprised of streptonigrin and 17-amino-geldanamycin attached via a dipeptide-p-aminobenzyl-amine linker system

Cytotoxic agents streptonigrin and 17-amino-geldanamycin were linked to monoclonal antibodies (mAbs), forming antibody–drug conjugates (ADCs) for antigen-mediated targeting to cancer cells. The drugs were conjugated with a linker construct that is labile to lysosomal proteases and incorporates a valine-alanine-p-aminobenzyl (PAB)-amino linkage for direct attachment to the electron-deficient amine functional groups present in both drugs. The resulting ADCs release drug following internalization into antigen-positive cancer cells. The drug linkers were conjugated to mAbs cAC10 (anti-CD30) and h1F6 (anti-CD70) via alkylation of reduced interchain disulfides to give ADCs loaded with 4 drugs/mAb. The streptonigrin ADCs were potent and immunologically specific on a panel of cancer cell lines in vitro and in a Hodgkin lymphoma xenograft model. We conclude that streptonigrin ADCs are candidates for further research, and that the novel linker system used to make them is well-suited for the conjugation of cytotoxic agents containing electron-deficient amine functional groups.     

Development and properties of beta-glucuronide linkers for monoclonal antibody-drug conjugates

A beta-glucuronide-based linker for attaching cytotoxic agents to monoclonal antibodies (mAbs) was designed and evaluated. We employed the cytotoxic auristatin derivatives MMAE (1a) and MMAF (1b) and doxorubicin propyloxazoline (DPO, 2) to give the beta-glucuronide drug-linkers 9a, 9b, and 17, respectively. Cysteine-quenched derivatives of 9b and 17 were determined to be substrates for E. coli beta-glucuronidase, resulting in facile drug release. The beta-glucuronide MMAF drug-linker 9b was highly stable in rat plasma with an extrapolated half-life of 81 days. Each drug-linker when conjugated to mAbs c1F6 (anti-CD70) and cAC10 (anti-CD30) gave monomeric antibody-drug conjugates (ADCs) with as many as eight drugs per mAb and had high levels of immunologically specific cytotoxic activity on cancer cell lines. cAC10-9a displayed pronounced antitumor activity in a subcutaneous Karpas 299 lymphoma tumor model. A single dose treatment led to cures in all animals at the 0.5 mg/kg dose level and above, and the conjugate was well tolerated at 100 mg/kg. In mice with subcutaneous renal cell carcinoma xenografts, the MMAF conjugate c1F6-9b was tolerated at 25 mg/kg and efficacious at 0.75 mg/kg. These results demonstrate that the beta-glucuronide linker system is an effective strategy for targeting cytotoxic agents providing ADCs with high degrees of efficacy at well-tolerated doses.     

Minor groove binder antibody conjugates employing a water soluble beta-glucuronide linker

The minor groove binder beta-glucuronide drug-linker 3 was constructed from amino CBI 1 and determined to be a substrate for Escherichia coli beta-glucuronidase (EC 3.2.1.31), resulting in facile drug release. Compound 3 was conjugated to mAbs cAC10 (anti-CD30) and h1F6 (anti-CD70) to give antibody-drug conjugates (ADCs) with potencies comparable to that of free drug 1. The ADCs were largely monomeric at intermediate loading levels (4-5drug/mAb), in contrast to highly aggregated p-aminobenzylcarbamate dipeptide-based ADCs of 1 previously reported. Significant levels of immunologic specificity were observed with cAC10-3 by comparing antigen positive versus negative cell lines and binding versus non-binding control ADCs. The water soluble beta-glucuronide linker is stable in plasma and effectively delivers drugs to target cells leading to potent cytotoxic activities.     

Conformational analysis by NMR and molecular dynamics of adamantane-doxorubicin prodrugs and their assemblies with β-cyclodextrin: A focus on the design of platforms for controlled drug delivery

Nanoscale design and construction of affinity-based drug delivery systems (ADDS) is an active research area with enormous potential for the improvement of cancer treatment. For the therapeutic load of these ADDS, a promising strategy is the design of pH-sensitive prodrugs based on the construction of conjugates between adamantane and doxorubicin (Ad-Dox), which stands out as an excellent model system to obtain novel supramolecular materials. Construction of these prodrugs involves a modification of three zones of doxorubicin which in principle does not affect the action mechanism: the carbonyl group C13 (hydrazone linker), the primary alcohol neighboring the carbonyl (ester linker) and the 3′ amino group of daunosamine sugar (amide linker). These modifications are aimed to improve the efficacy and reduce the systemic toxicity of the drug chemotherapy by controlling its release in cancer cells. In this work, we performed 2D NMR experiments and molecular dynamics simulations to characterize the conformational changes of three constructed prodrugs. Our results demonstrated that ring A and the daunsamine sugar of the hydrazone and amide linkers conserve the half-chair state ⁹H₈, while the ester linker disrupts this conformation. Our study also showed that the hydrazone-linked compound (Ad-h-Dox) does not modify the conformation of the original drug and maintains cytotoxic activity. Moreover, the inclusion complex (IC) of Ad-h-Dox with β-cyclodextrin (βCD) generated a highly soluble platform in water, whereas the ester-linked compound (Ad-e-Dox) causes the loss of biological activity. This study proves that Ad-h-Dox prodrug can be an optimum prodrug and act as a building block for a more complex drug transport system.     

Differential killing of CD56-expressing cells by drug-conjugated human antibodies targeting membrane-distal and membrane-proximal non-overlapping epitopes

CD56 (NCAM, neural cell adhesion molecule) is over-expressed in many tumor types, including neuroblastoma, multiple myeloma, small cell lung cancer, ovarian cancer, acute myeloid leukemia, NK-T lymphoma, neuroendocrine cancer and pancreatic cancer. Using phage display, we identified 2 high-affinity anti-CD56 human monoclonal antibodies (mAbs), m900 and m906, which bound to spatially separated non-overlapping epitopes with similar affinity (equilibrium dissociation constant 2.9 and 4.5 nM, respectively). m900 bound to the membrane proximal fibronectin type III-like domains, whereas m906 bound to the N-terminal IgG-like domains. m906 induced significant down-regulation of CD56 in 4 neuroblastoma cell lines tested, while m900-induced downregulation of CD56 was much lower. Antibody-drug conjugates (ADCs) made by conjugation with a highly potent pyrrolobenzodiazepine dimer (PBD) exhibited killing activity that correlated with CD56 down-regulation, and to some extent with in vivo binding ability of the antibodies. The m906PBD ADC was much more potent than m900PBD, likely due to higher CD56-mediated downregulation and stronger binding to cells. Treatment with m906PBD ADC resulted in very potent cytotoxicity (IC50: 0.05–1.7 pM). These results suggest a novel approach for targeting CD56-expressing neuroblastoma cells. Further studies in animal models and in humans are needed to find whether these antibodies and their drug conjugates are promising candidate therapeutics.     

Acid-sensitive polyethylene glycol conjugates of doxorubicin: preparation, in vitro efficacy and intracellular distribution

Coupling anticancer drugs to synthetic polymers is a promising approach of enhancing the antitumor efficacy and reducing the side-effects of these agents. Doxorubicin maleimide derivatives containing an amide or acid-sensitive hydrazone linker were therefore coupled to alpha-methoxy-poly(ethylene glycol)-thiopropionic acid amide (MW 20000 Da), alpha,omega-bis-thiopropionic acid amide poly(ethylene glycol) (MW 20000 Da) or alpha-tert-butoxy-poly(ethylene glycol)-thiopropionic acid amide (MW 70000 Da) and the resulting polyethylene glycol (PEG) conjugates isolated through size-exclusion chromatography. The polymer drug derivatives were designed as to release doxorubicin inside the tumor cell by acid-cleavage of the hydrazone bond after uptake of the conjugate by endocytosis. The acid-sensitive PEG conjugates containing the carboxylic hydrazone bonds exhibited in vitro activity against human BXF T24 bladder carcinoma and LXFL 529L lung cancer cells with IC70 values in the range 0.02-1.5 microm (cell culture assay: propidium iodide fluorescence or colony forming assay). In contrast, PEG doxorubicin conjugates containing an amide bond between the drug and the polymer showed no in vitro activity. Fluorescence microscopy studies in LXFL 529 lung cancer cells revealed that free doxorubicin accumulates in the cell nucleus whereas doxorubicin of the acid-sensitive PEG doxorubicin conjugates is primarily localized in the cytoplasm. Nevertheless, the acid-sensitive PEG doxorubicin conjugates retain their ability to bind to calf thymus DNA as shown by fluorescence and visible spectroscopy studies. Results regarding the effect of an acid-sensitive PEG conjugate of molecular weight 20000 in the chorioallantoic membrane (CAM) assay indicate that this conjugate is significantly less embryotoxic than free doxorubicin although antiangiogenic effects were not observed.     

Secondary mAb--vcMMAE conjugates are highly sensitive reporters of antibody internalization via the lysosome pathway

Monoclonal antibodies (mAb) selectively recognizing tumor surface antigens are an important and evolving approach to targeted cancer therapy. One application of therapeutic mAbs is drug targeting via mAb-drug conjugate (ADC) technology. Identification of mAbs capable of internalizing following antigen binding has been accomplished by tracking decline of surface-bound mAb or by internalization of a secondary mAb linked to a toxin. These methods may not be sufficiently sensitive for screening nor wholly predictive of the mAbs' capacity for a specific drug delivery. We have developed a highly selective and sensitive method to detect mAbs for cell internalization and drug delivery. This system uses secondary anti-human or anti-murine mAbs conjugated to the high-potency drug monomethyl auristatin E (MMAE) via a highly stable, enzymatically cleavable linker. Prior studies of this drug linker technology demonstrated internalization of a primary ADC leads to trafficking to lysosomes, drug release by lysosomal cathepsin B, and ensuing cell death. A secondary antibody--drug conjugate (2 degrees ADC) capable of binding primary mAbs bound to the surface of antigen-positive cells has comparable drug delivery capability. The system is sufficiently sensitive to detect internalizing mAbs in nonclonal hybridoma supernatants and is predictive of the activity of subsequently produced primary ADC. Because of their high extracellular stability, the noninternalized 2 degrees ADC are 100--1000-fold less toxic to cells over extended periods of time, permitting an assay in which components can be added without need for separate wash steps. This homogeneous screening system is amenable to medium-throughput screening applications and enables the early identification of mAbs capable of intracellular trafficking for drug delivery and release.     

Enhanced activity of monomethylauristatin F through monoclonal antibody delivery: effects of linker technology on efficacy and toxicity

We have previously shown that antibody-drug conjugates (ADCs) consisting of cAC10 (anti-CD30) linked to the antimitotic agent monomethylauristatin E (MMAE) lead to potent in vitro and in vivo activities against antigen positive tumor models. MMAF is a new antimitotic auristatin derivative with a charged C-terminal phenylalanine residue that attenuates its cytotoxic activity compared to its uncharged counterpart, MMAE, most likely due to impaired intracellular access. In vitro cytotoxicity studies indicated that mAb-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-MMAF (mAb-L1-MMAF) conjugates were >2200-fold more potent than free MMAF on a large panel of CD30 positive hematologic cell lines. As with cAC10-L1-MMAE, the corresponding MMAF ADC induced cures and regressions of established xenograft tumors at well tolerated doses. To further optimize the ADC, several new linkers were generated in which various components within the L1 linker were either altered or deleted. One of the most promising linkers contained a noncleavable maleimidocaproyl (L4) spacer between the drug and the mAb. cAC10-L4-MMAF was approximately as potent in vitro as cAC10-L1-MMAF against a large panel of cell lines and was equally potent in vivo. Importantly, cAC10-L4-MMAF was tolerated at >3 times the MTD of cAC10-L1-MMAF. LCMS studies indicated that drug released from cAC10-L4-MMAF was the cysteine-L4-MMAF adduct, which likely arises from mAb degradation within the lysosomes of target cells. This new linker technology appears to be ideally suited for drugs that are both relatively cell-impermeable and tolerant of substitution with amino acids. Thus, alterations of the linker have pronounced impacts on toxicity and lead to new ADCs with greatly improved therapeutic indices.     

Novel cleavable cell‐penetrating peptide–drug conjugates: synthesis and characterization

We report the first drug conjugate with a negatively charged amphipathic cell‐penetrating peptide. Furthermore, we compare two different doxorubicin cell‐penetrating peptide conjugates, which are both unique in their properties, due to their net charge at physiological pH, namely the positively charged octaarginine and the negatively charged proline‐rich amphipathic peptide. These conjugates were prepared exploiting a novel heterobifunctional crosslinker to join the N‐terminal cysteine residue of the peptides with the aliphatic ketone of doxorubicin. This small linker contains an activated thiol as well as aminooxy functionality, capable of generating a stable oxime bond with the C‐13 carbonyl group of doxorubicin. The disulfide bond formed between the peptide and doxorubicin enables the release of the drug in the cytosol, as confirmed by drug‐release studies performed in the presence of glutathione. Additionally, the cytotoxicity as well as the cellular uptake and distribution of this tripartite drug delivery system was investigated in MCF‐7 and HT‐29 cell lines. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and in vitro antitumour activity of carboplatin analogues containing functional handles compatible for conjugation to drug delivery systems

We describe herein the synthesis of a series of carboplatin derivatives with different functional groups at position 3 of the cyclobutane ring. This pharmacomodulation approach aims at facilitating the vectorisation of these analogues, via their subsequent conjugation to a drug delivery system. Five different derivatives bearing a hydroxy, keto, iodo, azido or amino function at position 3 were synthesised. One of these compounds was coupled to a bifunctional maleimide-containing linker. All compounds were tested in vitro for their cytotoxicity on four different cell lines including two platinum-resistant colorectal cancer cell line (SK-OV-3, HCT116, D3E2, D5B7) using an MTS assay. Overall, the tested compounds were up to six times more potent than carboplatin, especially on D5B7 human colorectal cancer cells. We demonstrated that these modifications led to potent analogues which are compatible with conjugation to a drug delivery system.     

Synthesis and structure-activity relationship studies of 4,11-diaminonaphtho[2,3-f]indole-5,10-diones

We describe the synthesis of derivatives of 4,11-diaminonaphtho[2,3-f]indole-5,10-dione and their cytotoxicity for human tumor cells that express major determinants of altered anticancer drug response, the efflux pump P-glycoprotein, and non-functional p53. Nucleophilic substitution of methoxy groups in 4,11-dimethoxynaphtho[2,3-f]indole-5,10-dione with various ethylenediamines yielded the derivatives of 4,11-diaminonaphtho[2,3-f]indole-5,10-dione, the indole containing analogues of the antitumor agent ametantrone. The cytotoxicity of novel compounds for multidrug resistant, P-glycoprotein-expressing tumor cells is highly dependent on the N-substituent at the terminal amino group of the ethylenediamine moiety. Whereas p53 null colon carcinoma cells were less sensitive to the reference drug doxorubicin than their counterparts with wild type p53, the majority of novel naphthoindole derivatives were equally potent for both cell lines, regardless of the p53 status.     

Synthesis and Antiproliferative Activity of Conjugates of Anthracycline Antibiotics with Sesquiterpene Lactones of the Elecampane

The daunorubicin and doxorubicin anthracycline antibiotics were modified with the Inula helenium L. sesquiterpene lactones (alantolactone, isoalantolactone, and alloalantolactone) and with their epoxy derivatives. Antiproliferative properties of these conjugates were studied on tumor and normal cell lines. The daunorubicin conjugates with the sesquiterpene lactones (isoalantolactone, allantolactone, and alloalantolactone) and with their epoxy derivatives were found to exhibit the higher activity against human tumor cell lines than the corresponding doxorubicin conjugates. The daunorubicin conjugate with epoxyisoalantolactone proved to be the most effective compound, because it was more cytotoxic than daunorubicin towards a number of cell lines, including those daunorubicin-resistant, and did not affect normal human cells.     

Design,synthesis and evaluation of anti-CD123 antibody drug conjugates

Leukemia stem cells (LSCs) account for the development of drug resistance and increased recurrence rate in acute myeloid leukemia (AML) patients. Targeted drug delivery to leukemia stem cells remains a major challenge in AML chemotherapy. Overexpressed interleukin-3 receptor alpha chain, CD123, on the surface of leukemia stem cells was reported to be a potential target in AML treatment. Here, we designed and developed an antibody drug conjugate (CD123-CPT) by integrating anti-CD123 antibody with a chemotherapeutic agent, Camptothecin (CPT), via a disulfide linker. The linker is biodegradable in the presence of Glutathione (GSH, an endogenous component in cells), which leads to release of CPT. Anti-CD123 antibody conjugates showed significant higher cellular uptake in CD123-overexpressed tumor cells. More importantly, CD123-CPT demonstrated potent inhibitory effects on CD123-overexpressed tumor cells. Consequently, these results provide a promising targeted chemotherapeutical strategy for AML treatment.     

Structure-activity relationship study of arylsulfonylimidazolidinones as anticancer agents

In an effort to find novel N-arylsulfonylimidazolidinones as highly potent anticancer agent, the structure-activity relationship of ethyl 2-methyl-4-(2-oxo-4-phenylimidazolidin-1-ylsulfonyl)phenylcarbamate was explored through synthesis and evaluation of in vitro cytotoxicity of its analogs against HCT116, A549 and NCL-H460 cancer cell lines. Among the synthesized derivatives, the carbamate analogs (4a-f and 4k-p) exhibited superior cytotoxicity to doxorubicin for all cancer cell lines. The SAR studies of these derivatives confirm that the intact 4-phenyl-l-benzenesulfonylimidazolidinone has a pivotal role as a basic pharmacophore and hydrophobic substitutions only at 2-position of 1-aminobenzenesulfonyl moiety are beneficial for the enhancement of the activity.     

Development of potent monoclonal antibody auristatin conjugates for cancer therapy

We describe the in vitro and in vivo properties of monoclonal antibody (mAb)-drug conjugates consisting of the potent synthetic dolastatin 10 analogs auristatin E (AE) and monomethylauristatin E (MMAE), linked to the chimeric mAbs cBR96 (specific to Lewis Y on carcinomas) and cAC10 (specific to CD30 on hematological malignancies). The linkers used for conjugate formation included an acid-labile hydrazone and protease-sensitive dipeptides, leading to uniformly substituted conjugates that efficiently released active drug in the lysosomes of antigen-positive (Ag+) tumor cells. The peptide-linked mAb-valine-citrulline-MMAE and mAb-phenylalanine-lysine-MMAE conjugates were much more stable in buffers and plasma than the conjugates of mAb and the hydrazone of 5-benzoylvaleric acid-AE ester (AEVB). As a result, the mAb-Val-Cit-MMAE conjugates exhibited greater in vitro specificity and lower in vivo toxicity than corresponding hydrazone conjugates. In vivo studies demonstrated that the peptide-linked conjugates induced regressions and cures of established tumor xenografts with therapeutic indices as high as 60-fold. These conjugates illustrate the importance of linker technology, drug potency and conjugation methodology in developing safe and efficacious mAb-drug conjugates for cancer therapy.     

Synthesis and biological activity of conjugates between paclitaxel and the cell delivery vector penetratin

Synthesis of paclitaxel-penetratin (pAntp) constructs, in which the 2'- or 7-position of paclitaxel was used as the attachment site for linker connecting the drug and peptide moieties, is described. Paclitaxel-2'-pAntp[43-58]-NH(2)3b and paclitaxel-2'-pAntp[52-58]-NH(2)3c showed excellent antitumour activity against human lung and breast cancer cell lines. These conjugates were highly soluble and stable with a half-life of >8h under cell culture conditions. The drug-peptide conjugates may be therapeutically useful due to improved pharmaceutical properties.     

Synthesis and biological evaluation of PSMA-targeting paclitaxel conjugates

Prostate cancer (PC) is the second most commonly occurring cancer in men. Conventional chemotherapy has wide variety of disadvantages such as high systemic toxicity and low selectivity. Targeted drug delivery is a promising approach to decrease side effects of therapy. Prostate specific membrane antigen (PSMA) is overexpressed in prostate cancer cells while low level of expression is observed in normal cells. In this study we describe the development of Glu-urea-Lys based PSMA-targeting conjugates with paclitaxel. A series of new PSMA targeting conjugates with paclitaxel was designed and synthesized. The cytotoxicity of conjugates was evaluated against prostate (LNCaP, 22Rv1 and PC-3) and non-prostate (Hek293T, VA13, A549 and MCF-7) cell lines. The most promising conjugate 21 was examined in vivo using 22Rv1 xenograft mice model. It demonstrated good efficiency comparable with paclitaxel, while reduced toxicity. 3D molecular docking study was also performed to understand underlying mechanism of binding and further optimization of the linker substructure and conjugates structure for improving the target affinity. These conjugates may be useful for further design of novel PSMA targeting delivery systems for PC.     

Preparation and functional evaluation of new doxorubicin immunoconjugates containing an acid-sensitive linker on small-cell lung cancer cells

 The anthracycline doxorubicin (DOX) is one of the most effective drugs for the treatment of small-cell lung cancer (SCLC), but its clinical application is limited by unspecific side-effects like cardiotoxicity. In the present study doxorubicin was conjugated to the monoclonal antibodies (mAb) SEN7, MOC31, and SWA11 via a novel acid-sensitive hydrazone linker. These mAb recognize SCLC-associated antigens of cluster 1 (NCAM), cluster 2 (EGP-2/GA733-2), and cluster 4 (CD24) respectively. To assess their potential therapeutic use against SCLC, the antigen-binding activities, the rates of internalization and the cytotoxic effects of the immunoconjugates were examined on tumour cell lines. The preparation procedure preserved the antigen-binding activities of the mAb and yielded immunoconjugates with average drug : mAb ratios of 7 : 1. The hydrazone linker was found to be stable at neutral pH but to release doxorubicin under acidic conditions. In contrast to SEN7-DOX, MOC31-DOX and SWA11-DOX were rapidly internalized into SCLC target cells upon binding to their specific cell-surface antigens. Accordingly, both immunoconjugates proved to be highly cytotoxic agents, inhibiting thymidine incorporation by 50% at concentrations between 0.5 μM and 1 μM and were 100-fold more selective than free doxorubicin. The results suggest that binding to selective cell-surface antigens, rapid internalization and efficient release of doxorubicin from the mAb by acid hydrolysis are required for the selective and potent function of the immunoconjugates. In particular, the use of MOC31-DOX for targeted cytotoxic therapy might be promising because of the limited cross-reactivity of the mAb with normal human tissues and its recently demonstrated tumour localization potential in SCLC patients. Received: 22 September 1995 / Accepted: 23 November 1995     

Antibody–drug conjugates as novel anti-cancer chemotherapeutics

Over the past couple of decades, antibody–drug conjugates (ADCs) have revolutionized the field of cancer chemotherapy. Unlike conventional treatments that damage healthy tissues upon dose escalation, ADCs utilize monoclonal antibodies (mAbs) to specifically bind tumour-associated target antigens and deliver a highly potent cytotoxic agent. The synergistic combination of mAbs conjugated to small-molecule chemotherapeutics, via a stable linker, has given rise to an extremely efficacious class of anti-cancer drugs with an already large and rapidly growing clinical pipeline. The primary objective of this paper is to review current knowledge and latest developments in the field of ADCs. Upon intravenous administration, ADCs bind to their target antigens and are internalized through receptor-mediated endocytosis. This facilitates the subsequent release of the cytotoxin, which eventually leads to apoptotic cell death of the cancer cell. The three components of ADCs (mAb, linker and cytotoxin) affect the efficacy and toxicity of the conjugate. Optimizing each one, while enhancing the functionality of the ADC as a whole, has been one of the major considerations of ADC design and development. In addition to these, the choice of clinically relevant targets and the position and number of linkages have also been the key determinants of ADC efficacy. The only marketed ADCs, brentuximab vedotin and trastuzumab emtansine (T-DM1), have demonstrated their use against both haematological and solid malignancies respectively. The success of future ADCs relies on improving target selection, increasing cytotoxin potency, developing innovative linkers and overcoming drug resistance. As more research is conducted to tackle these issues, ADCs are likely to become part of the future of targeted cancer therapeutics.     

Novel peptide linkers for highly potent antibody-auristatin conjugate

Auristatins are highly potent antimitotic agents that have received considerable attention because of their activities when targeted to tumor cells in the form of antibody-drug conjugates (ADCs). Our lead agent, SGN-35, consists of the cAC10 antibody linked to the N-terminal amino acid of monomethylauristatin E (MMAE) via a valine-citrulline p-aminobenzylcarbamate (val-cit-PABC) linker that is cleaved by intracellular proteases such as cathepsin B. More recently, we developed an auristatin F (AF) derivative monomethylauristatin F (MMAF), which unlike MMAE contains the amino acid phenylalanine at the C-terminal position. Because of the negatively charged C-terminal residue, the potency of AF and MMAF is impaired. However, their ability to kill target cells is greatly enhanced through facilitated cellular uptake by internalizing mAbs. Here, we explore the effects of linker technology on AF-based ADC potency, activity, and tolerability by generating a diverse set of dipeptide linkers between the C-terminal residue and the mAb carrier. The resulting ADCs differed widely in activity, with some having significantly improved therapeutic indices compared to the original mAb-Val-Cit-PABC-MMAF conjugate. The therapeutic index was increased yet further by generating dipeptide-based ADCs utilizing new auristatins with methionine or tryptophan as the C-terminal drug residue. These results demonstrate that manipulation of the C-terminal peptide sequence used to attach auristatins to the mAb carrier can lead to highly potent and specific conjugates with greatly improved therapeutic windows.