Povetacicept, an Enhanced Dual APRIL/BAFF Antagonist That Modulates B Lymphocytes and Pathogenic Autoantibodies for the Treatment of Lupus and Other B Cell-Related Autoimmune Diseases.

OBJECTIVE: Dysregulated APRIL/BAFF signaling is implicated in the pathogenesis of multiple autoimmune diseases, including systemic lupus erythematosus and lupus nephritis. We undertook this study to develop and evaluate a high-affinity APRIL/BAFF antagonist to overcome the clinical limitations of existing B cell inhibitors. METHODS: A variant of TACI-Fc generated by directed evolution showed enhanced binding for both APRIL and BAFF and was designated povetacicept (ALPN-303). Povetacicept was compared to wild-type (WT) TACI-Fc and related molecules in vitro and in vivo. RESULTS: Povetacicept inhibited APRIL and BAFF more effectively than all evaluated forms of WT TACI-Fc and selective APRIL and BAFF inhibitors in cell-based reporter assays and primary human B cell assays, mediating potent suppression of B cell proliferation, differentiation, and immunoglobulin (Ig) secretion. In mouse immunization models, povetacicept significantly reduced serum immunoglobulin titers and antibody-secreting cells more effectively than anti-CD20 monoclonal antibodies, WT TACI-Fc, or APRIL and BAFF inhibitors. In the NZB × NZW mouse lupus nephritis model, povetacicept significantly enhanced survival and suppressed proteinuria, anti-double-stranded DNA antibody titers, blood urea nitrogen, glomerulonephritis, and renal immunoglobulin deposition. In the bm12 mouse lupus model, povetacicept significantly reduced splenic plasmablasts, follicular helper T cells, and germinal center B cells. In non-human primates, povetacicept was well tolerated, exhibited high serum exposure, and significantly decreased serum IgM, IgA, and IgG levels after a single dose. CONCLUSION: Enhanced APRIL and BAFF inhibition by povetacicept led to greater inhibition of B cell populations critical for autoantibody production compared to WT TACI-Fc and CD20-, APRIL-, or BAFF-selective inhibitors. Potent, dual inhibition by povetacicept has the potential to significantly improve clinical outcomes in autoantibody-related autoimmune diseases.

First-in-human study of the safety, tolerability, pharmacokinetics, and pharmacodynamics of ALPN-101, a dual CD28/ICOS antagonist, in healthy adult subjects.

ALPN-101 (ICOSL vIgD-Fc) is an Fc fusion protein of a human inducible T cell costimulatory ligand (ICOSL) variant immunoglobulin domain (vIgD) designed to inhibit the cluster of differentiation 28 (CD28) and inducible T cell costimulator (ICOS) pathways simultaneously. A first-in-human study evaluated the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of ALPN-101 in healthy adult subjects. ALPN-101 was generally well-tolerated with no evidence of cytokine release, clinically significant immunogenicity, or severe adverse events following single subcutaneous (SC) doses up to 3 mg/kg or single intravenous (IV) doses up to 10 mg/kg or up to 4 weekly IV doses of up to 1 mg/kg. ALPN-101 exhibited a dose-dependent increase in exposure with an estimated terminal half-life of 4.3-8.6 days and SC bioavailability of 60.6% at 3 mg/kg. Minimal to modest accumulation in exposure was observed with repeated IV dosing. ALPN-101 resulted in a dose-dependent increase in maximum target saturation and duration of high-level target saturation. Consistent with its mechanism of action, ALPN-101 inhibited cytokine production in whole blood stimulated by Staphylococcus aureus enterotoxin B ex vivo, as well as antibody responses to keyhole limpet hemocyanin immunization, reflecting immunomodulatory effects upon T cell and T-dependent B cell responses, respectively. In conclusion, ALPN-101 was well-tolerated in healthy subjects with dose-dependent PK and PD consistent with the known biology of the CD28 and ICOS costimulatory pathways. Further clinical development of ALPN-101 in inflammatory and/or autoimmune diseases is therefore warranted.

Antibody-conjugated drug assay for protease-cleavable antibody-drug conjugates.

BACKGROUND: Antibody-drug conjugates (ADCs) require multiple assays to characterize their PK. These assays can separately evaluate the ADC by quantifying the antibody or the conjugated drug and may give different answers due to assay measurement differences, heterogeneous nature of ADCs and potential biotransformations that occur in vivo. RESULTS: We present a new version of the antibody-conjugated drug assay for valine-citrulline-linked monomethylauristatin E (vcMMAE) ADCs. A stable isotope-labeled internal standard, protein A affinity capture and solid-phase cleavage of MMAE using papain was used prior to LC-MS/MS analysis. CONCLUSION: The assay was used to assess the difference in ex vivo drug-linker stability of native-cysteine versus engineered cysteine ADCs and to determine the number of drugs per antibody of a native-cysteine ADC in vivo.

A potent anti-CD70 antibody-drug conjugate combining a dimeric pyrrolobenzodiazepine drug with site-specific conjugation technology.

A highly cytotoxic DNA cross-linking pyrrolobenzodiazepine (PBD) dimer with a valine-alanine dipeptide linker was conjugated to the anti-CD70 h1F6 mAb either through endogenous interchain cysteines or, site-specifically, through engineered cysteines at position 239 of the heavy chains. The h1F6239C-PBD conjugation strategy proved to be superior to interchain cysteine conjugation, affording an antibody-drug conjugate (ADC) with high uniformity in drug-loading and low levels of aggregation. In vitro cytotoxicity experiments demonstrated that the h1F6239C-PBD was potent and immunologically specific on CD70-positive renal cell carcinoma (RCC) and non-Hodgkin lymphoma (NHL) cell lines. The conjugate was resistant to drug loss in plasma and in circulation, and had a pharmacokinetic profile closely matching that of the parental h1F6239C antibody capped with N-ethylmaleimide (NEM). Evaluation in CD70-positive RCC and NHL mouse xenograft models showed pronounced antitumor activities at single or weekly doses as low as 0.1 mg/kg of ADC. The ADC was tolerated at 2.5 mg/kg. These results demonstrate that PBDs can be effectively used for antibody-targeted therapy.

Intracellular activation of SGN-35, a potent anti-CD30 antibody-drug conjugate.

PURPOSE: SGN-35 is an antibody-drug conjugate (ADC) containing the potent antimitotic drug, monomethylauristatin E (MMAE), linked to the anti-CD30 monoclonal antibody, cAC10. As previously shown, SGN-35 treatment regresses and cures established Hodgkin lymphoma and anaplastic large cell lymphoma xenografts. Recently, the ADC has been shown to possess pronounced activity in clinical trials. Here, we investigate the molecular basis for the activities of SGN-35 by determining the extent of targeted intracellular drug release and retention, and bystander activities. EXPERIMENTAL DESIGN: SGN-35 was prepared with (14)C-labeled MMAE. Intracellular ADC activation on CD30(+) and negative cell lines was determined using a combination of radiometric and liquid chromatograhpy/mass spectrometry-based assays. The bystander activity of SGN-35 was determined using mixed tumor cell cultures consisting of CD30(+) and CD30(-) lines. RESULTS: SGN-35 treatment of CD30(+) cells leads to efficient intracellular release of chemically unmodified MMAE, with intracellular concentrations of MMAE in the range of 500 nmol/L. This was due to specific ADC binding, uptake, MMAE retention, and receptor recycling or resynthesis. MMAE accounts for the total detectable released drug from CD30(+) cells, and has a half-life of retention of 15 to 20 h. Cytotoxicity studies with mixtures of CD30(+) and CD30(-) cell lines indicated that diffusible released MMAE from CD30(+) cells was able to kill cocultivated CD30(-) cells. CONCLUSIONS: MMAE is efficiently released from SGN-35 within CD30(+) cancer cells and, due to its membrane permeability, is able to exert cytotoxic activity on bystander cells. This provides mechanistic insight into the pronounced preclinical and clinical antitumor activities observed with SGN-35.

Contribution of linker stability to the activities of anticancer immunoconjugates.

The linker component of antibody-drug conjugates (ADC) is a key feature in developing optimized therapeutic agents that are highly active at well tolerated doses. For maximal intratumoral drug delivery, linkers are required that are highly stable in the systemic circulation, yet allow for efficient drug release at the target site. In this respect, amide bond-based technologies constitute a technological advancement, since the linker half-lives in circulation ( t 1/2 approximately 7 days) are much longer than earlier generation linkers that break down within 1-2 days. The amide linkers, some of which contain peptides, are appended to the mAb carriers through thioether/maleimide adducts. Here, we describe that use of a bromoacetamidecaproyl (bac) in place of the maleimidocaproyl (mc) increases the plasma stability of resulting thioether ADCs. One such ADC, 1F6-C4v2-bac-MMAF, which is directed against the CD70 antigen on lymphomas and renal cell carcinoma, was prepared containing a bac thioether spacer between the drug (MMAF) and the mAb carrier (1F6-C4v2). There was no measurable systemic drug release from this ADC for 2 weeks postadministration in mice. In order to assess the impact of improving linker stability beyond mc containing ADCs, a series of mc and bac-linked 1F6-MMAF conjugates were compared for tolerability, intratumoral drug delivery, and therapeutic efficacy in nude mice with renal cell carcinoma xenografts. There were no statistically significant efficacy differences between sets of mc and bac containing ADCs, although the bac linker technology led to 25% higher intratumoral drug exposure over a 7 day period compared to the corresponding mc linker. The mechanism of drug release from maleimide-adducts likely involves a retro-Michael reaction that takes place in plasma, based on in vitro studies demonstrating that some of the released drug-maleimide derivative became covalently bound to cysteine-34 of serum albumin. In summary, the data indicate that new linkers can be obtained with improved in vivo stability by replacing the maleimide with an acetamide, but the resulting ADCs had similar tolerability and activity profiles.

Lysosomal trafficking and cysteine protease metabolism confer target-specific cytotoxicity by peptide-linked anti-CD30-auristatin conjugates.

The chimeric anti-CD30 monoclonal antibody cAC10, linked to the antimitotic agents monomethyl auristatin E (MMAE) or F (MMAF), produces potent and highly CD30-selective anti-tumor activity in vitro and in vivo. These drugs are appended via a valine-citrulline (vc) dipeptide linkage designed for high stability in serum and conditional cleavage and putative release of fully active drugs by lysosomal cathepsins. To characterize the biochemical processes leading to effective drug delivery, we examined the intracellular trafficking, internalization, and metabolism of the parent antibody and two antibody-drug conjugates, cAC10vc-MMAE and cAC10vc-MMAF, following CD30 surface antigen interaction with target cells. Both cAC10 and its conjugates bound to target cells and internalized in a similar manner. Subcellular fractionation and immunofluorescence studies demonstrated that the antibody and antibody-drug conjugates entering target cells migrated to the lysosomes. Trafficking of both species was blocked by inhibitors of clathrin-mediated endocytosis, suggesting that drug conjugation does not alter the fate of antibody-antigen complexes. Incubation of cAC10vc-MMAE or cAC10vc-MMAF with purified cathepsin B or with enriched lysosomal fractions prepared by subcellular fractionation resulted in the release of active, free drug. Cysteine protease inhibitors, but not aspartic or serine protease inhibitors, blocked antibody-drug conjugate metabolism and the ensuing cytotoxicity of target cells and yielded enhanced intracellular levels of the intact conjugates. These findings suggest that in addition to trafficking to the lysosomes, cathepsin B and perhaps other lysosomal cysteine proteases are requisite for drug release and provide a mechanistic basis for developing antibody-drug conjugates cleavable by intracellular proteases for the targeted delivery of anti-cancer therapeutics.

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.

In vivo drug-linker stability of an anti-CD30 dipeptide-linked auristatin immunoconjugate.

Effective antibody-drug conjugates (ADC) combine high drug-linker stability in circulation and efficient intratumoral release of drug. Conjugation of monomethyl auristatin E (MMAE) to the anti-CD30 monoclonal antibody (mAb), cAC10, produced a selective and potent ADC against CD30(+) anaplastic large cell lymphoma and Hodgkin's disease models. This ADC, cAC10-valine-citrulline-MMAE, uses a protease-sensitive dipeptide linker designed to release MMAE by lysosomal cathepsin B in target cells but maintain a stable linkage and attenuate drug potency in circulation. To evaluate ADC stability in vivo, we developed methods for measuring drug/mAb ratios at progressive times in plasma from ADC-treated mice and nonhuman primates. Anti-idiotype mAb permitted the capture and quantitation of mAb cAC10, whereas antidrug mAb and MMAE-conjugated horseradish peroxidase reporter provided quantitative detection of conjugated drug following its in vitro release by cathepsin B. These data were validated by an alternative ELISA using anti-idiotype and anti-MMAE mAbs for capture and detection, respectively. Both methods differentiated ADC with variable levels of drug loading and were subsequently applied to stability studies in severe combined immunodeficient mice and cynomolgus monkeys. Evaluation of ADC from mouse circulation showed the linker half-life to be approximately 144 hours (6.0 days), significantly greater than that reported for disulfide- or hydrazone-linked ADCs in mice or human trials. In cynomolgus monkey, the apparent linker half-life was approximately 230 hours (9.6 days), suggesting that the drug-linker will be highly stable in humans. These data represent the longest reported drug-linker half-life to date and provide the basis for the pronounced specificity and antitumor activity of cAC10-valine-citrulline-MMAE.

Down-regulation of DNA repair synthesis at DNA single-strand interruptions in poly(ADP-ribose) polymerase-1 deficient murine cell extracts.

The functional involvement of poly(ADP-ribose) polymerase-1 (PARP-1) in the repair of DNA single- and double-strand breaks, DNA base damage, and related repair substrate intermediates remains unclear. Using an in vitro DNA repair assay and cell extracts derived from PARP-1 deficient or wild-type murine embryonic fibroblasts, we investigated the DNA synthesis and ligation steps associated with the rejoining of DNA single-strand interruptions containing 3'-OH, and either 5'-OH or 5'-P termini. Complete repair leading to DNA rejoining was similar between PARP-1 deficient cells and wild-type controls and poly(ADP-ribose) synthesis was, as expected, greatly reduced in PARP-1 deficient cell extracts. The incorporation of [32P]dCMP into repaired DNA at the site of a lesion was reduced two-three-fold in PARP-1 deficient cell extracts, demonstrating a decrease in repair patch size. Addition of purified PARP-1 to levels approximating those present in wild-type extracts did not stimulate DNA repair synthesis. We conclude that PARP-1 is not required for the efficient processing and rejoining of single-strand interruptions with defined 3'-OH and 5'-OH or 5'-P termini. Decreased DNA repair synthesis observed in PARP-1 deficient cell extracts is associated with reduced cellular expression of several factors required for long-patch base excision repair (BER), including FEN-1 and DNA ligase I.