Antibody analytics and sequences.

Pharmacokinetics of BCMA bispecific and ADC in cynomolgus monkeys.

Abstract 4–1BB (CD137) is an activation-induced costimulatory receptor that regulates immune responses of activated CD8 T and natural killer cells, by enhancing proliferation, survival, cytolytic activity, and IFNγ production. The ability to induce potent antitumor activity by stimulating 4–1BB on tumor-specific cytotoxic T cells makes 4–1BB an attractive target for designing novel immuno-oncology therapeutics. To minimize systemic immune toxicities and enhance activity at the tumor site, we have developed a novel bispecific antibody that stimulates 4–1BB function when co-engaged with the tumor-associated antigen 5T4. ALG.APV-527 was built on the basis of the ADAPTIR bispecific platform with optimized binding domains to 4–1BB and 5T4 originating from the ALLIGATOR-GOLD human single-chain variable fragment library. The epitope of ALG.APV-527 was determined to be located at domain 1 and 2 on 4–1BB using X-ray crystallography. As shown in reporter and primary cell assays in vitro, ALG.APV-527 triggers dose-dependent 4–1BB activity mediated only by 5T4 crosslinking. In vivo, ALG.APV-527 demonstrates robust antitumor responses, by inhibiting growth of established tumors expressing human 5T4 followed by a long-lasting memory immune response. ALG.APV-527 has an antibody-like half-life in cynomolgus macaques and was well tolerated at 50.5 mg/kg. ALG.APV-527 is uniquely designed for 5T4-conditional 4–1BB–mediated antitumor activity with potential to minimize systemic immune activation and hepatotoxicity while providing efficacious tumor-specific responses in a range of 5T4-expressing tumor indications as shown by robust activity in preclinical in vitro and in vivo models. On the basis of the combined preclinical dataset, ALG.APV-527 has potential as a promising anticancer therapeutic for the treatment of 5T4-expressing tumors.

Tables show PK kinetics, plasma cell numbers, and patient data. Methods include all supplemental methods.

T cell dynamics in response to BCMA bispecific and ADCs in cynomolgus monkeys.

BCMA bispecific is highly active in vivo.

BCMA cleavable and non-cleavable ADCs are highly active against xenograft tumors.

BCMA bispecific is activity in the presence of soluble BCMA and APRIL.

Background 4-1BB (CD137) is an activation-induced co-stimulatory receptor that regulates immune responses of activated CD8+ T cells and NK cells. Leveraging the therapeutic benefit of 1st generation 4-1BB monospecifics has been challenging due to dose limiting hepatotoxicity. To minimize systemic immune toxicities and enhance activity at the tumor site, we have developed a novel 4-1BB x 5T4 bispecific antibody that stimulates 4-1BB function only when co-engaged with 5T4, a highly selective tumor-associated antigen. The combined preclinical dataset presented here provides an overview of the potential indication landscape, mechanism of action and the efficacy and safety profile of ALG.APV-527, supporting its advancement into the clinic. Methods Genevestigator Software was used to analyze curated transcriptomic data from bulk tumor mRNA-sequencing data libraries and from single cell RNA-seq libraries for the expression profiles of CD8, 4-1BB and 5T4 across selected human solid tumor datasets. ADCC and ADCP reporter bioassays were utilized to assess Fc engagement by ALG.APV-527. For in vitro tumor lysis studies, human T cells were co-cultured with labelled tumor cells and sub-optimally activated with anti-CD3. Cytotoxicity of tumor cells were continually assessed using a Live-Cell Analysis System. Results Dual expression of CD8 and 5T4 occurred in many tumor types and correlated well with indications that are pursued in the clinical development of ALG.APV-527. 4-1BB expression was observed in tumor-derived lymphoid subpopulations, especially in those with an exhausted phenotype. Since ALG.APV-527 is designed with a non-Fcγ receptor binding Fc, minimal ADCC & ADCP was induced in vitro. Additionally, ALG.APV-527 enhanced primary immune cell-mediated killing of 5T4-expressing tumor cells when compared to anti-CD3 alone, demonstrating the potential benefit of 4-1BB agonism for enhancing cytotoxic anti-tumor responses in the clinic. Conclusions ALG. APV-527 is designed to elicit safe and efficacious 4-1BB-mediated antitumor activity in a range of 5T4-expressing tumor indications. Transcriptional profiling of patient tumor samples demonstrates 4-1BB expression in multiple tumor-infiltrating lymphocyte subsets and identifies potential indications with 5T4 expression and CD8+ T cell infiltration. The unique design of the molecule minimizes systemic immune activation and hepatotoxicity, allowing for highly efficacious tumor-specific responses as demonstrated by potent activity in in vitro models. Based on these preclinical data, ALG.APV-527 is a promising anti-cancer therapeutic for the treatment of a variety of 5T4-expressing solid tumors and is progressing towards a phase I clinical trial in 2021.

Abstract APVO603 is Aptevo's bispecific candidate targeting 4-1BB and OX40. It was designed to have unique properties with the potential to overcome some of the clinical challenges observed with monoclonal antibody targeting these receptors. APVO603 is engineered as an FcγR-signaling deficient bispecific antibody that utilizes Aptevo's ADAPTIR technology for a distinct approach for dual targeting of 4-1BB and OX40 in the absence of additional effector activity. The distinct characteristics of APVO603 may enable conditional activation of 4-1BB and OX40 via agonism of these receptors only when cross-linked via engagement of the other receptor via cis and/or trans cellular interactions. Thus, APVO603 is designed with the potential to overcome both the on-target toxicity and limited efficacy observed with 4-1BB and OX40 monoclonal antibody treatment in the clinic. Anti-4-1BB and OX40 binding domains were optimized to increase affinity, function and stability, then incorporated into the ADAPTIR bispecific antibody platform to produce the APVO603 lead candidate. APVO603 was found to augment 4-1BB and OX40 activity in a dose-dependent manner and is strictly dependent on engagement of the reciprocal receptor to elicit 4-1BB or OX40 signaling in vitro. In preclinical assays using PBMCs sub-optimally stimulated with anti-CD3, APVO603 induces synergistic proliferation of CD4+, CD8+ T and NK cells when compared to anti-OX40 or 4-1BB antibodies with a wt Fc, included either individually or in combination. Additionally, APVO603 enhances proinflammatory cytokine production, granzyme B expression, and reduces the T cell exhaustion phenotype. The mechanistic activity of APVO603 resulted in dose-dependent control of in vivo tumor growth in a preclinical humanized murine xenograft model using established murine MB49 bladder tumors in human 4-1BB and OX40 double knock-in mice. APVO603 is a dual-agonistic bispecific antibody that augments the effector function of activated CD4+ and CD8+ T cells and NK cells in a dose-dependent manner and reduces growth of established tumors in vivo. This preclinical data demonstrates conditional dual stimulation of 4-1BB and OX40 and supports further development of APVO603, a promising immuno-oncology therapeutic with potential for benefit in solid tumors. This program is progressing into IND-enabling studies later this year. Citation Format: Michelle H. Nelson, Robert E. Miller, Secil Franke-Welch, Ruth Chenault, Hang Fang, Allison Chunyk, Gabriela Hernandez-Hoyos, Hilario J. Ramos, David Bienvenue, Catherine McMahan. APVO603: A dual 4-1BB and OX40 bispecific approach utilizing ADAPTIRTMtechnology designed to deliver a conditional T cell/NK response against solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB173.

Abstract Prostate-Specific Membrane Antigen (PSMA), is a tumor-associated antigen (TAA) that is expressed on prostate cancers, including metastatic castration-resistant prostate cancer (mCRPC). Current chemotherapeutic approaches for mCRPC are challenged by development of resistance resulting in limited clinical benefit. Immuno-oncology therapeutic candidates such as bispecifics re-directing T-cell responses to eliminate tumors, are a promising strategy to overcome the limitations of current approaches and provide benefit to patients with aggressive cancers. Here we present preclinical data demonstrating a potential new approach using low affinity targeting of CD3 and high affinity targeting of PSMA for treatment of a solid tumor cancer.APVO442 is Aptevo's bispecific candidate targeting PSMA and CD3. This candidate was designed to have unique pharmacokinetic and safety properties to potentially maximize potent anti-tumor responses against mCRPC. The APVO442 bispecific T-cell engager uses Aptevo's ADAPTIR-FLEX technology to generate a molecule with low-affinity monovalent CD3 engagement, paired with high-affinity bivalent PSMA binding designed to deliver a highly selective T-cell response at the tumor. The unique engineering of APVO442 reduces the potential of binding to CD3 expressed on peripheral T cells, thus minimizing the potential for on-target toxicity, such as cytokine release, and increasing the potential to deliver the effective concentration of the molecule localized to solid tumors.Preclinical testing demonstrated that APVO442 exhibits optimal manufacturability and functional characteristics for lead candidate selection. Anti-PSMA x anti-CD3 constructs with varying binding strengths to CD3 were evaluated for specificity of CD3 binding, ability to enhance T-cell activation, and ability to elicit T-cell-mediated cytotoxicity against PSMA-expressing tumor targets with varying levels of PSMA expression. APVO442 demonstrated 10-fold reduced binding to CD3 and EC50 compared to the highest affinity constructs tested while retaining equivalent binding to tumor cells expressing various levels of PSMA. The differences in CD3 affinity were associated in a slightly lower EC50 of potency however, the maximal responses for in vitro activation of CD4+ and CD8+ T cells including upregulation of CD25/CD69 expression, proliferation and in vitro tumor lysis were comparable between low and high affinity CD3 constructs. Finally, APVO442 induced reduced levels of multiple cytokines in vitro when compared to high affinity competitor molecules.In vivo, APVO442 elicited robust anti-tumor responses of human PSMA-expressing tumors in a murine xenograft tumor model. The in vivo activity of APVO442 was comparable to high affinity CD3 engaging comparators with similarly measured PK profiles. Additional in vivo characterization of APVO442 is ongoing and continued pre-clinical studies are planned for 2021. Citation Format: Rebecca Gottschalk, Robert E. Miller, Lynda Misher, Michelle H. Nelson, Allison Chunyk, Brian Woodruff, Elizabeth Haglin, Gabriela Hernandez-Hoyos, Peter Pavlik, Catherine McMahan, Hilario J. Ramos, David Bienvenue. APVO442: A bispecific T cell-engaging candidate utilizing the ADAPTIR-FLEXTMplatform technology with unique properties designed for optimal tumor distribution and cytotoxic response against PSMA-expressing solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB172.

Background4-1BB (CD137) is an activation-induced co-stimulatory receptor that regulates immune responses of activated CD8+ T cells and NK cells, by enhancing proliferation, survival, cytolytic activity and IFN-γ production. Its ability to induce potent anti-tumor CD8+ and NK cell activity makes 4-1BB an attractive target for designing novel therapeutics for immuno-oncology. However, clinical development of a monospecific 4-1BB agonistic antibody has been hampered by dose-limiting hepatic toxicities. To minimize systemic immune toxicities and enhance activity at the tumor site, we have developed a novel 4-1BB x 5T4 bispecific antibody that stimulates 4-1BB function only when co-engaged with 5T4, a tumor-associated antigen. The combined preclinical dataset presented here provides an overview of the mechanism of action and the efficacy and safety profile of ALG.APV-527, supporting its advancement into the clinic.MethodsALG. APV-527 was built based the ADAPTIR™ platform with binding domains to 4-1BB and 5T4 generated using the ALLIGATOR-GOLD® human scFv library. ALG.APV-527 was tested using primary cells in the presence or absence of cells expressing 5T4. Cell Trace-labelled PBMC sub-optimally stimulated with anti-CD3, to induce 4-1BB expression, cells were gated using flow cytometry. T cell cytotoxicity was assessed by quantifying cell death in CD8+ T cell/tumor cell co-cultures, and images were obtained using a cell live imaging system (Cytation 5). For tumor inhibition studies, human 4-1BB knock-in mice were injected subcutaneously with MB49 cells transfected with human 5T4. Cured mice were subsequently used in a toxicity study and liver pathology was evaluated.ResultsIn vitro, ALG.APV-527 enhances primary CD8+ T cell and NK cell function and proliferation in the presence of 5T4-expressing cells. Using imaging, ALG.APV-527 in combination with a bispecific T cell engager caused increased cell death in T cell/tumor cell co-cultures. ALG.APV-527 inhibited growth of established tumors at doses as low as 2 µg/mouse in a syngeneic bladder cancer model. Following recovery, mice exhibited a memory response when rechallenged with tumor. In a high dose safety study in human 4-1BB knock-in mice, ALG.APV-527 did not cause significant systemic immune activation, whereas urelumab analogue treated mice induced dermatitis, elevated serum cytokines, CD8+ T-cell liver infiltration and systemic T-cell proliferation.ConclusionsALG. APV-527 induces potent CD8+ T cell and NK cell co-stimulation and T-cell cytotoxicity and has potent in vivo anti-tumor activity, without inducing systemic toxicity. Based on preclinical data, ALG.APV-527 is a promising anti-cancer therapeutic for the treatment of a variety of 5T4-expressing solid tumors.Ethics ApprovalAll studies were review and approved by the Internal Animal Care and Use Committee (IACUC) of Aptevo Therapeutics

Abstract The restricted expression pattern of B-cell maturation antigen (BCMA) makes it an ideal tumor-associated antigen (TAA) for the treatment of myeloma. BCMA has been targeted by both CD3 bispecific antibody and antibody–drug conjugate (ADC) modalities, but a true comparison of modalities has yet to be performed. Here we utilized a single BCMA antibody to develop and characterize both a CD3 bispecific and 2 ADC formats (cleavable and noncleavable) and compared activity both in vitro and in vivo with the aim of generating an optimal therapeutic. Antibody affinity, but not epitope was influential in drug activity and hence a high-affinity BCMA antibody was selected. Both the bispecific and ADCs were potent in vitro and in vivo, causing dose-dependent cell killing of myeloma cell lines and tumor regression in orthotopic myeloma xenograft models. Primary patient cells were effectively lysed by both CD3 bispecific and ADCs, with the bispecific demonstrating improved potency, maximal cell killing, and consistency across patients. Safety was evaluated in cynomolgus monkey toxicity studies and both modalities were active based on on-target elimination of B lineage cells. Distinct nonclinical toxicity profiles were seen for the bispecific and ADC modalities. When taken together, results from this comparison of BCMA CD3 bispecific and ADC modalities suggest better efficacy and an improved toxicity profile might be achieved with the bispecific modality. This led to the advancement of a bispecific candidate into phase I clinical trials.

Monoclonal antibody therapies are an important approach for the treatment of hematologic malignancies, but typically show low single‐agent activity. Bispecific antibodies, however, redirect immune cells to the tumor for subsequent lysis, and preclinical and accruing clinical data support single‐agent efficacy of these agents in hematologic malignancies, presaging an exciting era in the development of novel bispecific formats. This review discusses recent developments in this area, highlighting the challenges in delivering effective immunotherapies for patients.