Recombinant human IgG antibodies (hIgGs) completely devoid of binding to Fcγ receptors (FcγRs) and complement protein C1q, and thus with abolished immune effector functions, are of use for various therapeutic applications in order to reduce FcγR activation and Fc-mediated toxicity. Fc engineering approaches described to date only partially achieve this goal or employ a large number of mutations, which may increase the risk of anti-drug antibody generation. We describe here two new, engineered hIgG Fc domains, hIgG1-P329G LALA and hIgG4-P329G SPLE, with completely abolished FcγR and C1q interactions, containing a limited number of mutations and with unaffected FcRn interactions and Fc stability. Both 'effector-silent' Fc variants are based on a novel Fc mutation, P329G that disrupts the formation of a proline sandwich motif with the FcγRs. As this motif is present in the interface of all IgG Fc/FcγR complexes, its disruption can be applied to all human and most of the other mammalian IgG subclasses in order to create effector silent IgG molecules.
Dysregulated cellular apoptosis and resistance to cell death are hallmarks of neoplastic initiation and disease progression. Therefore, the development of agents that overcome apoptosis dysregulation in tumor cells is an attractive therapeutic approach. Activation of the extrinsic apoptotic pathway is strongly dependent on death receptor (DR) hyperclustering on the cell surface. However, strategies to activate DR5 or DR4 through agonistic antibodies have had only limited clinical success. To pursue an alternative approach for tumor-targeted induction of apoptosis, we engineered a bispecific antibody (BsAb), which simultaneously targets fibroblast-activation protein (FAP) on cancerassociated fibroblasts in tumor stroma and DR5 on tumor cells. We hypothesized that bivalent binding to both FAP and DR5 leads to avidity-driven hyperclustering of DR5 and subsequently strong induction of apoptosis in tumor cells but not in normal cells.Here, we show that RG7386, an optimized FAP-DR5 BsAb, triggers potent tumor cell apoptosis in vitro and in vivo in preclinical tumor models with FAP-positive stroma. RG7386 antitumor efficacy was strictly FAP dependent, was independent of FcR crosslinking, and was superior to conventional DR5 antibodies. In combination with irinotecan or doxorubicin, FAP-DR5 treatment resulted in substantial tumor regression in patient-derived xenograft models. FAP-DR5 also demonstrated single-agent activity against FAP-expressing malignant cells, due to cross-binding of FAP and DR5 across tumor cells. Taken together, these data demonstrate that RG7386, a novel and potent antitumor agent in both mono-and combination therapies, overcomes limitations of previous DR5 antibodies and represents a promising approach to conquer tumor-associated resistance to apoptosis.
Background Obinutuzumab (GA101) is a third generation, glycoengineered defucosylated anti-CD20 antibody which shows higher binding affinity for CD16A than fully glycosylated rituximab (RTX). This higher binding leads to stronger NK mediated antibody dependent cellular cytotoxicity activity (ADCC) by obinutuzumab compared to that induced by RTX. The GPI-anchored CD16B molecule is highly homologous to CD16A and is the major FcγR on polymorphonuclear neutrophils (PMN). We have therefore investigated the binding of obinutuzumab to CD16B and its functional activity on human PMN compared to parent rituximab (RTX) or to defucosylated rituximab (G2 antibody). Methods Binding to CD16B of glycoengineered or fully glycosylated anti-CD20 antibodies was measured by surface plasmon resonance (Biacore). For PMN activation and phagocytosis, we have used either purified PMN or analyzed PMN function in unmanipulated whole blood assays from normal donors or CLL patients. PMN activation was measured as CD11b upregulation and CD62L downmodulation by flow cytometry. Phagocytosis by PMN of chronic lymphocytic leukemia (CLL) cells was measured by triple fluorescence (PKH26, CD15-FITC and CD19-APC) and flow cytometry. Results Obinutuzumab or glycoengineered defucosylated rituximab (called G2) bound CD16B with about 7 fold higher affinity, compared to non-glycoengineered wild type parental antibodies. This was true either using surface plasmon resonance or measuring antibody binding to live PMN. Furthermore obinutuzumab activated PMN, either purified or in whole blood, more efficiently than RTX. Activation resulted in a 50% increase in CD11b expression and 70% down-modulation of CD62L on PMN and in release of TNFα, IL-6 and IL-8. Activation was not accompanied by generation of reactive oxygen species or ADCC, but led to phagocytosis of anti-CD20 antibody opsonized CLL targets by purified PMN. Indeed up to 50% phagocytic PMN could be observed in presence of obinutuzumab or G2 antibodies after 6-24 hours incubation of purified PMN with CLL targets. Significant phagocytosis (15%) was also observed in whole blood, but only in presence of glycoengineered antibodies, and was followed by up to 50% PMN death. Finally we show, using blocking F(ab) and F(ab’)2 fragments specific for CD16B and CD32A, that both these receptors are involved in PMN activation, phagocytosis and cell death induced by glycoengineered anti-CD20 antibodies. The possible effect of NA1 and NA2 polymorphisms of CD16B on obinutuzumab binding and phagocytosis is under further investigation. Conclusions We conclude that phagocytosis by PMN is an additional mechanism of action of obinutuzumab, mediated through its higher binding affinity for CD16B compared to RTX. Phagocytosis takes place in whole blood and is followed by PMN death. This effect may in part explain the neutropenia observed after treatment of B-CLL patients with GA101. Disclosures: Golay: Roche Glycart AG: Research Funding. Ferrara Koller:Roche Glycart AG: Employment. Rambaldi:Roche Italia: Consultancy, Honoraria. Klein:Roche Glycart AG: Employment. Introna:Roche Glycart AG: Research Funding.
Biotherapeutics may contain a multitude of different post-translational modifications (PTMs) that need to be assessed and possibly monitored and controlled to ensure reproducible product quality. During early development of biotherapeutics, unexpected PTMs might be prevented by in silico identification and characterization together with further molecular engineering. Mass determinations of a human IgG1 (mAb1) and a bispecific IgG-ligand fusion protein (BsAbA) demonstrated the presence of unusual PTMs resulting in major +80 Da, and +16/+32 Da chain variants, respectively. For mAb1, analytical cation exchange chromatography demonstrated the presence of an acidic peak accounting for 20%. A + 79.957 Da modification was localized within the light chain complementarity-determining region-2 and identified as a sulfation based on accurate mass, isotopic distribution, and a complete neutral loss reaction upon collision-induced dissociation. Top-down ultrahigh resolution MALDI-ISD FT-ICR MS of modified and unmodified Fabs allowed the allocation of the sulfation to a specific Tyr residue. An aspartate in amino-terminal position-3 relative to the affected Tyr was found to play a key role in determining the sulfation. For BsAbA, a + 15.995 Da modification was observed and localized to three specific Pro residues explaining the +16 Da chain A, and +16 Da and +32 Da chain B variants. The BsAbA modifications were verified as 4-hydroxyproline and not 3-hydroxyproline in a tryptic peptide map via cochromatography with synthetic peptides containing the two isomeric forms. Finally, our approach for an alert system based on in-house in silico predictors is presented. This system is designed to prevent these PTMs by molecular design and engineering during early biotherapeutic development.
Synthetic T cell redirecting therapies, using chimeric antigen receptor (CAR)-T cells or CD3-bispecific antibodies targeting B-cell surface antigens such as CD19 and CD20, currently in clinical development, are emerging as promising, potential therapeutic approaches for the treatment of non-Hodgkin lymphomas (NHL). CD3-bispecific antibodies and first generation CAR-T cells only provide T cell receptor stimulation, so-called "signal 1", to the redirected T cells, but lack costimulatory, so-called "signal 2", support of those T cells. Agonism of costimulatory receptors on T cells, such as CD28 and/or 4-1BB, can increase the strength and durability of a T cell-mediated response via multiple mechanisms. Co-stimulation can enhance T cell specific cytotoxicity, proliferation, secretion of Th1-polarizing cytokines, recruitment of additional T cells via increased chemokine secretion, T cell metabolic fitness, and resistance to T-cell exhaustion and to activation-induced T-cell death. Indeed, 2nd generation CAR-T cells that incorporate CD28 or 4-1BB co-stimulation have replaced 1st generation ones in clinical development. However, complex manufacturing logistics and the need of specialized clinical centers for the administration of CAR-T cells significantly limit their broad application. In order to provide an off-the-shelf, synthetic T cell redirection approach delivering both signals 1 and 2 to T cells, CD3-bispecific antibodies would need combination with systemically administered T-cell costimulatory agonists. Yet, clinical development of 1st generation costimulatory agonists has not been successful to date due to on-target, off-tumor immune-mediated toxicity, such as hepatotoxicity. To overcome this limitation, we have generated a novel 4-1BB costimulatory agonist, CD19-targeted 4-1BBL (CD19-4-1BBL, RG6076, RO7227166), and are developing it in combination with a potent CD20xCD3 T cell bispecific antibody, CD20-TCB (RG6026 or glofitamab). CD19-4-1BBL consists of a trimeric, human 4-1BBL fused to a monovalent CD19-targeting IgG1 antibody with an engineered Fc region devoid of FcgR binding. As effective agonism of 4-1BB receptor requires crosslinking of more than three receptor units on a T cell, CD19-4-1BBL is systemically inactive unless it binds to CD19 and clusters on the surface of targeted B-cells to hyper-crosslink multiple 4-1BB receptors on redirected T cells. In our off-the-shelf, combination approach, glofitamab binds to CD20 on B-cells and engages CD3 on redirected T cells, providing signal 1 and inducing the expression of 4-1BB on those T cells. CD19-4-1BBL can then target those activated T cells and provide them with signal 2. In preclinical experiments, we show that CD19-4-1BBL can boost glofitamab-mediated cytokine release by activated T cells in healthy donor as well as DLBCL patient-derived PBMCs. Using a human diffuse large B cell lymphoma (DLBCL) tumor-bearing (WSU-DLCL2) fully humanized mouse model, we observed a CD19-4-1BBL dose-dependent, synergistic combination effect with glofitamab, leading to strongly increased T cell accumulation in tumors, tumor growth inhibition and regression. Importantly, CD19-4-1BBL was also able to prevent tumor escape to glofitamab monotherapy at late treatment time points in a fully humanized mouse model bearing large OCI-Ly18 human DLBCL tumors. Glofitamab monotherapy has recently demonstrated encouraging activity in relapsed/refractory NHL patients with reported complete response rates in DLBCL in the same range as those of 2nd generation CAR-T cells that already incorporate both T cell signals 1 and 2. The preclinical data we report here provide a strong rationale for adding CD19-4-1BBL-mediated T cell signal 2 to glofitamab in the clinic to further boost treatment efficacy and deliver an off-the-shelf, enhanced T cell redirection approach alternative to CAR-T cell therapy. CD19-4-1BBL is currently in clinical trials (NCT04077723). Disclosures Herter: Roche Glycart AG:Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.Sam:Roche Glycart AG:Current Employment.Ferrara Koller:Roche Glycart AG:Current Employment.Diggelmann:Roche Glycart AG:Current Employment, Current equity holder in publicly-traded company.Bommer:Roche Glycart AG:Current Employment.Schönle:Roche Glycart AG:Current Employment.Claus:Roche Glycart AG:Current Employment.Bacac:Roche Glycart AG:Current Employment, Patents & Royalties.Klein:Roche:Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.Umana:Roche Glycart AG:Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.
<p>Figure S2. Related to Figure 3. In vitro characterization of the FAP-DR5 BsAb RG7386; Table S4. Characterization of RG7386 binding avidities.</p>
<p>Supplementary information on cell maintenance, and antibody selection and characterization</p>
<p>Characterization and optimization of RG7386 dosing in vivo.</p>
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