The HER2-directed antibody-drug conjugate DHES0815A in advanced and/or metastatic breast cancer: preclinical characterization and phase 1 trial results.

Approved antibody-drug conjugates (ADCs) for HER2-positive breast cancer include trastuzumab emtansine and trastuzumab deruxtecan. To develop a differentiated HER2 ADC, we chose an antibody that does not compete with trastuzumab or pertuzumab for binding, conjugated to a reduced potency PBD (pyrrolobenzodiazepine) dimer payload. PBDs are potent cytotoxic agents that alkylate and cross-link DNA. In our study, the PBD dimer is modified to alkylate, but not cross-link DNA. This HER2 ADC, DHES0815A, demonstrates in vivo efficacy in models of HER2-positive and HER2-low cancers and is well-tolerated in cynomolgus monkey safety studies. Mechanisms of action include induction of DNA damage and apoptosis, activity in non-dividing cells, and bystander activity. A dose-escalation study (ClinicalTrials.gov: NCT03451162) in patients with HER2-positive metastatic breast cancer, with the primary objective of evaluating the safety and tolerability of DHES0815A and secondary objectives of characterizing the pharmacokinetics, objective response rate, duration of response, and formation of anti-DHES0815A antibodies, is reported herein. Despite early signs of anti-tumor activity, patients at higher doses develop persistent, non-resolvable dermal, ocular, and pulmonary toxicities, which led to early termination of the phase 1 trial.

T cell-Dependent Bispecific Therapy Enhances Innate Immune Activation and Antibody-Mediated Killing.

T cell-retargeting therapies have transformed the therapeutic landscape for hematologic diseases. T cell-dependent bispecific antibodies (TDB) function as conditional agonists that induce a polyclonal T-cell response, resulting in target cell destruction and cytokine release. The relationship between this response and its effects on surrounding innate immune populations has not been fully explored. Here we show that treatment with mosunetuzumab in patients results in natural killer (NK) cell activation in the peripheral blood. We modeled this phenomenon in vitro and found that TDB-mediated killing activated NK cells, increasing NK function and antibody-dependent cellular cytotoxicity (ADCC), and enhanced the capability of macrophages to perform antibody-dependent cellular phagocytosis (ADCP). This enhancement was triggered by cytokines released through TDB treatment, with IL2 and IFNγ being major drivers for increased ADCC and ADCP, respectively. Surprisingly, cytolytic ability could be further augmented through neutralization of IL10 for NK cells and TNFα for macrophages. Finally, we showed that TDB treatment enhanced the efficacy of Fc-driven killing to an orthogonal solid tumor target in vivo. These results provide rationale for novel antibody therapy combinations that take advantage of both adaptive and innate immune responses.

Targeting MCL-1 and BCL-2 with polatuzumab vedotin and venetoclax overcomes treatment resistance in R/R non-Hodgkin lymphoma: Results from preclinical models and a Phase Ib study.

The treatment of patients with relapsed or refractory lymphoid neoplasms represents a significant clinical challenge. Here, we identify the pro-survival BCL-2 protein family member MCL-1 as a resistance factor for the BCL-2 inhibitor venetoclax in non-Hodgkin lymphoma (NHL) cell lines and primary NHL samples. Mechanistically, we show that the antibody-drug conjugate polatuzumab vedotin promotes MCL-1 degradation via the ubiquitin/proteasome system. This targeted MCL-1 antagonism, when combined with venetoclax and the anti-CD20 antibodies obinutuzumab or rituximab, results in tumor regressions in preclinical NHL models, which are sustained even off-treatment. In a Phase Ib clinical trial (NCT02611323) of heavily pre-treated patients with relapsed or refractory NHL, 25/33 (76%) patients with follicular lymphoma and 5/17 (29%) patients with diffuse large B-cell lymphoma achieved complete or partial responses with an acceptable safety profile when treated with the recommended Phase II dose of polatuzumab vedotin in combination with venetoclax and an anti-CD20 antibody.

Selective PROTAC-mediated degradation of SMARCA2 is efficacious in SMARCA4 mutant cancers.

The mammalian SWItch/Sucrose Non-Fermentable (SWI/SNF) helicase SMARCA4 is frequently mutated in cancer and inactivation results in a cellular dependence on its paralog, SMARCA2, thus making SMARCA2 an attractive synthetic lethal target. However, published data indicates that achieving a high degree of selective SMARCA2 inhibition is likely essential to afford an acceptable therapeutic index, and realizing this objective is challenging due to the homology with the SMARCA4 paralog. Herein we report the discovery of a potent and selective SMARCA2 proteolysis-targeting chimera molecule (PROTAC), A947. Selective SMARCA2 degradation is achieved in the absence of selective SMARCA2/4 PROTAC binding and translates to potent in vitro growth inhibition and in vivo efficacy in SMARCA4 mutant models, compared to wild type models. Global ubiquitin mapping and proteome profiling reveal no unexpected off-target degradation related to A947 treatment. Our study thus highlights the ability to transform a non-selective SMARCA2/4-binding ligand into a selective and efficacious in vivo SMARCA2-targeting PROTAC, and thereby provides a potential new therapeutic opportunity for patients whose tumors contain SMARCA4 mutations.

Development of T-cell engagers selective for cells co-expressing two antigens.

T cell-engaging bispecific antibodies (TCEs) are clinically effective treatments for hematological cancers. While the utility of TCEs in solid malignancies is being explored, toxicities arising from antigen expression on normal tissues have slowed or halted several clinical trials. Here, we describe the development of TCEs that preferentially drive T cell-mediated death against target cells co-expressing two tumor-associated antigens. We show that Ly6E and B7-H4 are simultaneously expressed on approximately 50% of breast cancers, whereas normal tissue expression is limited and mostly orthogonal. Traditional bispecific TCEs targeting a singular antigen, either Ly6E or B7-H4, are active when paired with high-affinity CD3-engagers, but normal tissue expression presents a toxicity risk. Treatment with a murine cross-reactive B7-H4-TCE results in rapid and severe weight loss in mice along with damage to B7-H4-expressing tissues. To overcome on-target toxicity, we designed trispecific antibodies co-targeting Ly6E, B7-H4, and CD3 and characterized the impact of dual-antigen binding and the relative placement of each binding domain on tumor killing in vitro and in vivo. In vitro killing of tumor cells co-expressing both antigens correlates to the placement of the higher affinity B7-H4 binding domain, with only modest enhancements seen upon addition of Ly6E binding. In xenograft models, avid binding of appropriately designed trispecific TCEs enables tumor growth inhibition while evading the poor tolerability seen with active bispecific TCEs. Collectively these data highlight the potential for dual-antigen targeting to improve safety and efficacy, and expand the scope of tumors that may effectively be treated by TCEs.Abbreviations: Chimeric antigen receptor T cells (CAR-Ts), dual-antigen targeted T cell engagers (DAT-TCE), Fragment antigen-binding (Fab), Hematoxylin and eosin (H&E), Institutional Animal Care and Use Committee (IACUC), Immunoglobulin G (IgG), immunohistochemistry (IHC), NOD SCID gamma (NSG), peripheral blood mononuclear cells (PBMCs), surface plasmon resonance (SPR), T cell-engagers (TCEs).

A homogeneous high-DAR antibody-drug conjugate platform combining THIOMAB antibodies and XTEN polypeptides.

The antibody-drug conjugate (ADC) is a well-validated modality for the cell-specific delivery of small molecules with impact expanding rapidly beyond their originally-intended purpose of treating cancer. However, antibody-mediated delivery (AMD) remains inefficient, limiting its applicability to targeting highly potent payloads to cells with high antigen expression. Maximizing the number of payloads delivered per antibody is one key way in which delivery efficiency can be improved, although this has been challenging to carry out; with few exceptions, increasing the drug-to-antibody ratio (DAR) above ∼4 typically destroys the biophysical properties and in vivo efficacy for ADCs. Herein, we describe the development of a novel bioconjugation platform combining cysteine-engineered (THIOMAB) antibodies and recombinant XTEN polypeptides for the unprecedented generation of homogeneous, stable "TXCs" with DAR of up to 18. Across three different bioactive payloads, we demonstrated improved AMD to tumors and Staphylococcus aureus bacteria for high-DAR TXCs relative to conventional low-DAR ADCs.

Valency of HER2 Targeting Antibodies Influences Tumor Cell Internalization and Penetration.

T-cell-dependent bispecific antibodies (TDB) have been a major advancement in the treatment of cancer, allowing for improved targeting and efficacy for large molecule therapeutics. TDBs are comprised of one arm targeting a surface antigen on a cancer cell and another targeting an engaging surface antigen on a cytotoxic T cell. To impart this function, the antibody must be in a bispecific format as opposed to the more conventional bivalent format. Through in vitro and in vivo studies, we sought to determine the impact of changing antibody valency on solid tumor distribution and catabolism. A bivalent anti-HER2 antibody exhibited higher catabolism than its full-length monovalent binding counterpart in vivo by both invasive tissue harvesting and noninvasive single photon emission computed tomography/X-ray computed tomography imaging despite similar systemic exposures for the two molecules. To determine what molecular factors drove in vivo distribution and uptake, we developed a mechanistic model for binding and catabolism of monovalent and bivalent HER2 antibodies in KPL4 cells. This model suggests that observed differences in cellular uptake of monovalent and bivalent antibodies are caused by the change in apparent affinity conferred by avidity as well as differences in internalization and degradation rates of receptor bound antibodies. To our knowledge, this is the first study to directly compare the targeting abilities of monovalent and bivalent full-length antibodies. These findings may inform diverse antibody therapeutic modalities, including T-cell-redirecting therapies and drug delivery strategies relying upon receptor internalization.

Calicheamicin Antibody-Drug Conjugates with Improved Properties.

Calicheamicin antibody-drug conjugates (ADCs) are effective therapeutics for leukemias with two recently approved in the United States: Mylotarg (gemtuzumab ozogamicin) targeting CD33 for acute myeloid leukemia and Besponsa (inotuzumab ozogamicin) targeting CD22 for acute lymphocytic leukemia. Both of these calicheamicin ADCs are heterogeneous, aggregation-prone, and have a shortened half-life due to the instability of the acid-sensitive hydrazone linker in circulation. We hypothesized that we could improve upon the heterogeneity, aggregation, and circulation stability of calicheamicin ADCs by directly attaching the thiol of a reduced calicheamicin to an engineered cysteine on the antibody via a disulfide bond to generate a linkerless and traceless conjugate. We report herein that the resulting homogeneous conjugates possess minimal aggregation and display high in vivo stability with 50% of the drug remaining conjugated to the antibody after 21 days. Furthermore, these calicheamicin ADCs are highly efficacious in mouse models of both solid tumor (HER2+ breast cancer) and hematologic malignancies (CD22+ non-Hodgkin lymphoma). Safety studies in rats with this novel calicheamicin ADC revealed an increased tolerability compared with that reported for Mylotarg. Overall, we demonstrate that applying novel linker chemistry with site-specific conjugation affords an improved, next-generation calicheamicin ADC.

Antibody-Mediated Delivery of Chimeric BRD4 Degraders. Part 2: Improvement of In Vitro Antiproliferation Activity and In Vivo Antitumor Efficacy.

Heterobifunctional compounds that direct the ubiquitination of intracellular proteins in a targeted manner via co-opted ubiquitin ligases have enormous potential to transform the field of medicinal chemistry. These chimeric molecules, often termed proteolysis-targeting chimeras (PROTACs) in the chemical literature, enable the controlled degradation of specific proteins via their direction to the cellular proteasome. In this report, we describe the second phase of our research focused on exploring antibody-drug conjugates (ADCs), which incorporate BRD4-targeting chimeric degrader entities. We employ a new BRD4-binding fragment in the construction of the chimeric ADC payloads that is significantly more potent than the corresponding entity utilized in our initial studies. The resulting BRD4-degrader antibody conjugates exhibit potent and antigen-dependent BRD4 degradation and antiproliferation activities in cell-based experiments. Multiple ADCs bearing chimeric BRD4-degrader payloads also exhibit strong, antigen-dependent antitumor efficacy in mouse xenograft assessments that employ several different tumor models.

An Anti-CD22-seco-CBI-Dimer Antibody-Drug Conjugate (ADC) for the Treatment of Non-Hodgkin Lymphoma That Provides a Longer Duration of Response than Auristatin-Based ADCs in Preclinical Models.

We are interested in developing a second generation of antibody-drug conjugates (ADCs) for the treatment of non-Hodgkin lymphoma (NHL) that could provide a longer duration of response and be more effective in indolent NHL than the microtubule-inhibiting ADCs pinatuzumab vedotin [anti-CD22-vc-monomethyl auristatin E (MMAE)] and polatuzumab vedotin (anti-CD79b-vc-MMAE). Pinatuzumab vedotin (anti-CD22-vc-MMAE) and polatuzumab vedotin (anti-CD79b-vc-MMAE) are ADCs that contain the microtubule inhibitor MMAE. Clinical trial data suggest that these ADCs have promising efficacy for the treatment of NHL; however, some patients do not respond or become resistant to the ADCs. We tested an anti-CD22 ADC with a seco-CBI-dimer payload, thio-Hu anti-CD22-(LC:K149C)-SN36248, and compared it with pinatuzumab vedotin for its efficacy and duration of response in xenograft models and its ability to deplete normal B cells in cynomolgus monkeys. We found that anti-CD22-(LC:K149C)-SN36248 was effective in xenograft models resistant to pinatuzumab vedotin, gave a longer duration of response, had a different mechanism of resistance, and was able to deplete normal B cells better than pinatuzumab vedotin. These studies provide evidence that anti-CD22-(LC:K149C)-SN36248 has the potential for longer duration of response and more efficacy in indolent NHL than MMAE ADCs and may provide the opportunity to improve outcomes for patients with NHL.

Preclinical optimization of Ly6E-targeted ADCs for increased durability and efficacy of anti-tumor response.

Early success with brentuximab vedotin in treating classical Hodgkin lymphoma spurred an influx of at least 20 monomethyl auristatin E (MMAE) antibody-drug conjugates (ADCs) into clinical trials. While three MMAE-ADCs have been approved, most of these conjugates are no longer being investigated in clinical trials. Some auristatin conjugates show limited or no efficacy at tolerated doses, but even for drugs driving initial remissions, tumor regrowth and metastasis often rapidly occur. Here we describe the development of second-generation therapeutic ADCs targeting Lymphocyte antigen 6E (Ly6E) where the tubulin polymerization inhibitor MMAE (Compound 1) is replaced with DNA-damaging agents intended to drive increased durability of response. Comparison of a seco-cyclopropyl benzoindol-4-one (CBI)-dimer (compound 2) to MMAE showed increased potency, activity across more cell lines, and resistance to efflux by P-glycoprotein, a drug transporter commonly upregulated in tumors. Both anti-Ly6E-CBI and -MMAE conjugates drove single-dose efficacy in xenograft and patient-derived xenograft models, but seco-CBI-dimer conjugates showed reduced tumor outgrowth following multiple weeks of treatment, suggesting that they are less susceptible to developing resistance. In parallel, we explored approaches to optimize the targeting antibody. In contrast to immunization with recombinant Ly6E or Ly6E DNA, immunization with virus-like particles generated a high-affinity anti-Ly6E antibody. Conjugates to this antibody improve efficacy versus a previous clinical candidate both in vitro and in vivo with multiple cytotoxics. Conjugation of compound 2 to the second-generation antibody results in a substantially improved ADC with promising preclinical efficacy.

Systematic Variation of Pyrrolobenzodiazepine (PBD)-Dimer Payload Physicochemical Properties Impacts Efficacy and Tolerability of the Corresponding Antibody-Drug Conjugates.

Cytotoxic pyrrolobenzodiazepine (PBD)-dimer molecules are frequently utilized as payloads for antibody-drug conjugates (ADCs), and many examples are currently in clinical development. In order to further explore this ADC payload class, the physicochemical properties of various PBD-dimer molecules were modified by the systematic introduction of acidic and basic moieties into their chemical structures. The impact of these changes on DNA binding, cell membrane permeability, and in vitro antiproliferation potency was, respectively, determined using a DNA alkylation assay, PAMPA assessments, and cell-based cytotoxicity measurements conducted with a variety of cancer lines. The modified PBD-dimer compounds were subsequently incorporated into CD22-targeting ADCs, and these entities were profiled in a variety of in vitro and in vivo experiments. The introduction of a strongly basic moiety into the PBD-dimer scaffold afforded a conjugate with dramatically worsened mouse tolerability properties relative to ADCs derived from related payloads, which lacked the basic group.

Effect of Modulating FcRn Binding on Direct and Pretargeted Tumor Uptake of Full-length Antibodies.

Full-length antibodies lack ideal pharmacokinetic properties for rapid targeted imaging, prompting the pursuit of smaller peptides and fragments. Nevertheless, studying the disposition properties of antibody-based imaging agents can provide critical insight into the pharmacology of their therapeutic counterparts, particularly for those coupled with potent payloads. Here, we evaluate modulation of binding to the neonatal Fc receptor (FcRn) as a protein engineering-based pharmacologic strategy to minimize the overall blood pool background with directly labeled antibodies and undesirable systemic click reaction of radiolabeled tetrazine with circulating pretargeted trans-cyclooctene (TCO)-modified antibodies. Noninvasive SPECT imaging of mice bearing HER2-expressing xenografts was performed both directly (111In-labeled antibody) and indirectly (pretargeted TCO-modified antibody followed by 111In-labeled tetrazine). Pharmacokinetic modulation of antibodies was achieved by two distinct methods: Fc engineering to reduce binding affinity to FcRn, and delayed administration of an antibody that competes with binding to FcRn. Tumor imaging with directly labeled antibodies was feasible in the absence of FcRn binding, rapidly attaining high tumor-to-blood ratios, but accompanied by moderate liver and spleen uptake. Pretargeted imaging of tumors with non-FcRn-binding antibody was also feasible, but systemic click reaction still occurred, albeit at lower levels than with parental antibody. Our findings demonstrate that FcRn binding impairment of full-length IgG antibodies moderately lowers tumor accumulation of radioactivity, and shifts background activity from blood pool to liver and spleen. Furthermore, reduction of FcRn binding did not eliminate systemic click reaction, but yielded greater improvements in tumor-to-blood ratio when imaging with directly labeled antibodies than with pretargeting.

Improved translation of stability for conjugated antibodies using an in vitro whole blood assay.

For antibody-drug conjugates to be efficacious and safe, they must be stable in circulation to carry the payload to the site of the targeted cell. Several components of a drug-conjugated antibody are known to influence stability: 1) the site of drug attachment on the antibody, 2) the linker used to attach the payload to the antibody, and 3) the payload itself. In order to support the design and optimization of a high volume of drug conjugates and avoid unstable conjugates prior to testing in animal models, we wanted to proactively identify these potential liabilities. Therefore, we sought to establish an in vitro screening method that best correlated with in vivo stability. While traditionally plasma has been used to assess in vitro stability, our evaluation using a variety of THIOMABTM antibody-drug conjugates revealed several disconnects between the stability assessed in vitro and the in vivo outcomes when using plasma. When drug conjugates were incubated in vitro for 24 h in mouse whole blood rather than plasma and then analyzed by affinity capture LC-MS, we found an improved correlation to in vivo stability with whole blood (R2 = 0.87, coefficient of determination) compared to unfrozen or frozen mouse plasma (R2 = 0.34, 0.01, respectively). We further showed that this whole blood assay was also able to predict in vivo stability of other preclinical species such as rat and cynomolgus monkey, as well as in human. The screening method utilized short (24 h) incubation times, as well as a custom analysis software, allowing increased throughput and in-depth biotransformation characterization. While some instabilities that were more challenging to identify remain, the method greatly enhanced the process of screening, optimizing, and lead candidate selection, resulting in the substantial reduction of animal studies.

Antibody Conjugation of a Chimeric BET Degrader Enables in†vivo Activity.

The ability to selectively degrade proteins with bifunctional small molecules has the potential to fundamentally alter therapy in a variety of diseases. However, the relatively large size of these chimeric molecules often results in challenging physico-chemical properties (e. g., low aqueous solubility) and poor pharmacokinetics which may complicate their in vivo applications. We recently discovered an exquisitely potent chimeric BET degrader (GNE-987) which exhibited picomolar cell potencies but also demonstrated low in vivo exposures. In an effort to improve the pharmacokinetic properties of this molecule, we discovered the first degrader-antibody conjugate by attaching GNE-987 to an anti-CLL1 antibody via a novel linker. A single IV dose of the conjugate afforded sustained in vivo exposures that resulted in antigen-specific tumor regressions. Enhancement of a chimeric protein degrader with poor in vivo properties through antibody conjugation thereby expands the utility of directed protein degradation as both a biological tool and a therapeutic possibility.

Biodistribution and efficacy of an anti-TENB2 antibody-drug conjugate in a patient-derived model of prostate cancer.

TENB2, a transmembrane proteoglycan protein, is a promising target for antibody drug conjugate (ADC) therapy due to overexpression in human prostate tumors and rapid internalization. We previously characterized how predosing with parental anti-TENB2 monoclonal antibody (mAb) at 1 mg/kg in a patient-derived LuCap77 explant model with high (3+) TENB2 expression could (i) block target-mediated intestinal uptake of tracer (& 0.1 mg/kg) levels of radiolabeled anti-TENB2-monomethyl auristatin E ADC while preserving tumor uptake, and (ii) maintain efficacy relative to ADC alone. Here, we systematically revisit this strategy to evaluate the effects of predosing on tumor uptake and efficacy in LuCap96.1, a low TENB2-expressing (1+) patient-derived model that is more responsive to ADC therapy than LuCap77. Importantly, rather than using tracer (& 0.1 mg/kg) levels, radiolabeled ADC tumor uptake was assessed at 1 mg/kg - one of the doses evaluated in the tumor growth inhibition study - in an effort to bridge tissue distribution (PK) with efficacy (PD). Predosing with mAb up to 1 mg/kg had no effect on efficacy. These findings warrant further investigations to determine whether predosing prior to ADC therapy might improve therapeutic index by preventing ADC disposition and possible toxicological liabilities in antigen-expressing healthy tissues.

Evaluation and use of an anti-cynomolgus monkey CD79b surrogate antibody-drug conjugate to enable clinical development of polatuzumab vedotin.

BACKGROUND AND PURPOSE: Polatuzumab vedotin is an antibody-drug conjugate (ADC) being developed for non-Hodgkin's lymphoma. It contains a humanized anti-CD79b IgG1 monoclonal antibody linked to monomethyl auristatin E (MMAE), an anti-mitotic agent. Polatuzumab vedotin binds to human CD79b only. Therefore, a surrogate ADC that binds to cynomolgus monkey CD79b was used to determine CD79b-mediated pharmacological effects in the monkey and to enable first-in-human clinical trials. EXPERIMENTAL APPROACH: Polatuzumab vedotin, the surrogate ADC, and the corresponding antibodies were evaluated in different assays in vitro and in animals. In vitro assessments included binding to peripheral blood mononuclear cells from different species, binding to a human and monkey CD79b-expressing cell line, binding to human Fcγ receptors, and stability in plasma across species. In vivo, ADCs were assessed for anti-tumour activity in mice, pharmacokinetics/pharmacodynamics in monkeys, and toxicity in rats and monkeys. KEY RESULTS: Polatuzumab vedotin and surrogate ADC bind with similar affinity to human and cynomolgus monkey B cells, respectively. Comparable in vitro plasma stability, in vivo anti-tumour activity, and mouse pharmacokinetics were also observed between the surrogate ADC and polatuzumab vedotin. In monkeys, only the surrogate ADC showed B-cell depletion and B-cell-mediated drug disposition, but both ADCs showed similar MMAE-driven myelotoxicity, as expected. CONCLUSIONS AND IMPLICATIONS: The suitability of the surrogate ADC for evaluation of CD79b-dependent pharmacology was demonstrated, and anti-tumour activity, pharmacokinetics/pharmacodynamics, and toxicity data with both ADCs supported the entry of polatuzumab vedotin into clinical trials.

Exposure-Efficacy Analysis of Antibody-Drug Conjugates Delivering an Excessive Level of Payload to Tissues.

Antibody-drug conjugates (ADCs) contain a disease-receptor antibody and a payload drug connected via a linker. The payload delivery depends on both tumor properties and ADC characteristics. In this study, we used different linkers, attachment sites, and doses to modulate payload delivery of several ADCs bearing maytansinoids (e.g., DM1), auristatins (e.g., MMAE), and DNA alkylating agents [e.g., pyrrolo[2,1-c][1,4]benzodiazepine-dimer (PBD)] as payloads in HER2- or CD22-expressing xenograft models. The tumor growth inhibition and ADC stability and exposure data were collected and analyzed from these dosed animals. The trend analysis suggests that intratumoral payload exposures that directly related the combination of conjugate linker and dose correlate with the corresponding efficacies of three payload types in two antigen-expressing xenograft models. These preliminary correlations also suggest that a minimal threshold concentration of intratumoral payload is required to support sustained efficacy. In addition, an ADC can deliver an excessive level of payload to tumors that does not enhance efficacy ("Plateau" effect). In contrast to tumor payload concentrations, the assessments of systemic exposures of total antibody (Tab) as well as the linker, dose, site of attachment, plasma stability, and drug-to-antibody ratio changes of these ADCs did not consistently rationalize the observed ADC efficacies. The requirement of a threshold payload concentration for efficacy is further supported by dose fractionation studies with DM1-, MMAE-, and PBD-containing ADCs, which demonstrated that single-dose regimens showed better efficacies than fractionated dosing. Overall, this study demonstrates that 1) the linker and dose together determine the tissue payload concentration that correlates with the antitumor efficacy of ADCs and 2) an ADC can deliver an unnecessary level of payload to tumors in xenograft models.

Antibody-Drug Conjugates Derived from Cytotoxic seco-CBI-Dimer Payloads Are Highly Efficacious in Xenograft Models and Form Protein Adducts In Vivo.

This work discloses the first examples of antibody-drug conjugates (ADCs) that are constructed from linker-drugs bearing dimeric seco-CBI payloads (duocarmycin analogs). Several homogeneous, CD22-targeting THIOMAB antibody-drug conjugates (TDCs) containing the dimeric seco-CBI entities are shown to be highly efficacious in the WSU-DLCL2 and BJAB mouse xenograft models. Surprisingly, the seco-CBI-containing conjugates are also observed to undergo significant biotransformation in vivo in mice, rats, and monkeys and thereby form 1:1 adducts with the Alpha-1-Microglobulin (A1M) plasma protein from these species. Variation of both the payload mAb attachment site and length of the linker-drug is shown to alter the rates of adduct formation. Subsequent experiments demonstrated that adduct formation attenuates the in vitro antiproliferation activity of the affected seco-CBI-dimer TDCs, but does not significantly impact the in vivo efficacy of the conjugates. In vitro assays employing phosphatase-treated whole blood suggest that A1M adduct formation is likely to occur if the seco-CBI-dimer TDCs are administered to humans. Importantly, protein adduct formation leads to the underestimation of total antibody (Tab) concentrations using an ELISA assay but does not affect Tab values determined via an orthogonal LC-MS/MS method. Several recommendations regarding bioanalysis of future in vivo studies involving related seco-CBI-containing ADCs are provided based on these collective findings.

Optimization of Pan-Pim Kinase Activity and Oral Bioavailability Leading to Diaminopyrazole (GDC-0339) for the Treatment of Multiple Myeloma.

Pim kinases have been targets of interest for a number of therapeutic areas. Evidence of durable single-agent efficacy in human clinical trials validated Pim kinase inhibition as a promising therapeutic approach for multiple myeloma patients. Here, we report the compound optimization leading to GDC-0339 (16), a potent, orally bioavailable, and well tolerated pan-Pim kinase inhibitor that proved efficacious in RPMI8226 and MM.1S human multiple myeloma xenograft mouse models and has been evaluated as an early development candidate.