Engineered Antibodies with Increased Activity to Recruit Complement

Idusogie, Esohe; Wong, Pin Yee; Presta, Leonard G.; Gazzano-Santoro, Hélène; Tótpál, Klára; Ultsch, Mark; Mulkerrin, Michael G.

doi:10.4049/jimmunol.166.4.2571

Cited by 266 publications

(175 citation statements)

References 23 publications

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“…5B), with Arg B129 acting like a wedge in between the CH2 and CL domains. This model is fully consistent with (i) the physicochemical characteristics of the ionic component of the C1q-IgG interaction (7), (ii) the mutagenesis experiments locating the C1q binding epicenter in human IgG1 around residues Asp 270 , Lys 322 , Pro 329 , Pro 331 (9,41,42), and (iii) the fact that the interaction involves two arginine residues of the C1q head (Arg B114 , Arg B129 ) proposed to mediate IgG recognition (44). The location of Tyr 278 at the interface with C1q (Fig.…”

Section: Discussionsupporting

confidence: 68%

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The Crystal Structure of the Globular Head of Complement Protein C1q Provides a Basis for Its Versatile Recognition Properties

Gaboriaud¹,

Juanhuix

Gruez³

et al. 2003

Journal of Biological Chemistry

323

366

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C1q is a versatile recognition protein that binds to an amazing variety of immune and non-immune ligands and triggers activation of the classical pathway of complement. The crystal structure of the C1q globular domain responsible for its recognition properties has now been solved and refined to 1.9 Å of resolution. The structure reveals a compact, almost spherical heterotrimeric assembly held together mainly by non-polar interactions, with a Ca 2؉ ion bound at the top. The heterotrimeric assembly of the C1q globular domain appears to be a key factor of the versatile recognition properties of this protein. Plausible three-dimensional models of the C1q globular domain in complex with two of its physiological ligands, C-reactive protein and IgG, are proposed, highlighting two of the possible recognition modes of C1q. The C1q/human IgG1 model suggests a critical role for the hinge region of IgG and for the relative orientation of its Fab domain in C1q binding.Innate immunity involves a combination of cell-surface receptors and soluble proteins with the ability to recognize microbial pathogens and thereby to generate signals that both orientate subsequent adaptive immune responses and trigger effector mechanisms (1, 2). Most of these molecules are oligomeric and recognize molecular patterns on microorganisms (3). An archetypal molecule of this type is C1q, the recognition subunit of C1, the complex that triggers activation of the classical pathway of complement, a major element of innate immunity. C1q is a 460-kDa protein with the overall shape of a bouquet of flowers, comprising six heterotrimeric collagen-like triple helices that associate in their N-terminal half to form a "stalk," then diverge to form individual "stems", each terminating in a C-terminal heterotrimeric globular domain (4). It is well documented that most of the C1 complex ligands are recognized by these peripheral globular domains, or heads, of C1q, thus triggering activation of C1r and C1s, the proteases associated with C1q (5). It is also established that C1q binds to immune complexes containing IgG or IgM, but not to those having IgA, IgD, or IgE (6). The major C1q binding site on IgG has been mapped to the CH2 domain of the Fc portion of the molecule (7-9). Although C1q shows marked differences in its reactivity toward IgG subclasses, the reason for this selectivity is not known.C1q is traditionally known for its ability to bind antibodies. However, it recognizes an amazing variety of other ligands. These include certain bacteria, viruses, parasites, and mycoplasma (6, 10 -12), underscoring its role as an antibody-independent defense protein. C1q also binds to C-reactive protein (CRP) 1 when complexed with exposed phosphocholine residues on bacteria, providing a further means of host defense (13). C1q is also capable of recognizing aberrant structures from self. Thus, in addition to cellular debris and sub-cellular membranes (14), it is established that C1q binds to, and induces clearance of, apoptotic cells (15), thereby playing a major role ...

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Section: Discussionsupporting

confidence: 68%

“…Interaction with IgG b12 was constrained by the location of the C1q binding site in human IgG1 as defined by mutagenesis data (9,41,42). The interaction between C1q and IgG is known to involve a major ionic component (7), and Lys 322 in hIgG1 has been identified by several groups as a key residue engaged in an ionic interaction with C1q (9,42).…”

Section: Methodsmentioning

confidence: 99%

The Crystal Structure of the Globular Head of Complement Protein C1q Provides a Basis for Its Versatile Recognition Properties

Gaboriaud¹,

Juanhuix

Gruez³

et al. 2003

Journal of Biological Chemistry

323

366

View full text Add to dashboard Cite

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“…The "knockout" and enhanced variants which we have generated exhibit desirable characteristics for therapeutic use. It will be interesting to know if our approach can be used in conjunction with a C H 2 engineering approach previously described (28) to further increase CDC activity. Further elucidation of the molecular mechanisms by which our hinge modifications modulate these functions will require a more detailed analysis, such as segmental flexibility measurements to experimentally verify the flexibility properties of the variants (5, 42), testing of alternate Fc␥RIIIA allotypes, generation of additional mutants and x-ray crystallography of IgG/C1q and IgG/Fc␥RIIIA complexes.…”

Section: Discussionmentioning

confidence: 99%

Modulation of the Effector Functions of a Human IgG1 through Engineering of Its Hinge Region

Dall’Acqua¹,

Cook²,

Damschroder³

et al. 2006

The Journal of Immunology

126

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We report here the engineering of a humanized anti-human EphA2 mAb (mAb 12G3H11) in an effort to explore the relationship between the hinge of a human IgG1 and its effector functions. mAb 12G3H11, used here as a model, is directed against the human receptor tyrosine kinase EphA2, which is an actively investigated target for cancer therapy due to its up-regulation in many cancer cells. Various rational modifications were introduced into the hinge region of mAb 12G3H11. These mutations were predicted to modulate the hinge’s length, flexibility, and/or biochemical properties. We show that the upper and middle hinge both play important, although functionally distinct roles. In particular, middle hinge modifications predicted to decrease its rigidity or length as well as eliminating either one of its two cysteine residues had a strong negative impact on C1q binding and complement-dependent cytotoxicity. Disruption of covalent bonds between both H chains may account in part for these effects. We also describe middle hinge mutants with a significantly decreased ability to bind FcγRIIIA and trigger Ab-dependent cell-mediated cytotoxicity. Conversely, we also generated upper hinge mutants exhibiting an increase in C1q binding and complement-dependent cytotoxicity activity. Therefore, this approach represents a novel strategy to fine-tune the biological activity of a given human IgG1. We also define, for the first time in such a systematic fashion, the relationship between various characteristics of the middle and upper hinge and the corresponding effector functions.

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“…Use of other anti-CD20 mAbs, which can kill B cells as F(abЈ) 2 , may provide an approach for targeting circulating CD20 ϩ cells in CLL (42). Alternatively, it should be possible to engineer RTX to activate complement, but not bind to Fc␥ receptors (48). Although such an engineered molecule may not be appropriate for the treatment of NHL, it may have therapeutic efficacy in CLL.…”

Section: Therapeutic Implicationsmentioning

confidence: 99%

Rituximab Infusion Promotes Rapid Complement Depletion and Acute CD20 Loss in Chronic Lymphocytic Leukemia

Kennedy

Beum

Solga

et al. 2004

The Journal of Immunology

321

340

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Complement plays an important role in the immunotherapeutic action of the anti-CD20 mAb rituximab, and therefore we investigated whether complement might be the limiting factor in rituximab therapy. Our in vitro studies indicate that at high cell densities, binding of rituximab to human CD20+ cells leads to loss of complement activity and consumption of component C2. Infusion of rituximab in chronic lymphocytic leukemia patients also depletes complement; sera of treated patients have reduced capacity to C3b opsonize and kill CD20+ cells unless supplemented with normal serum or component C2. Initiation of rituximab infusion in chronic lymphocytic leukemia patients leads to rapid clearance of CD20+ cells. However, substantial numbers of B cells, with significantly reduced levels of CD20, return to the bloodstream immediately after rituximab infusion. In addition, a mAb specific for the Fc region of rituximab does not bind to these recirculating cells, suggesting that the rituximab-opsonized cells were temporarily sequestered by the mononuclear phagocytic system, and then released back into the circulation after the rituximab-CD20 complexes were removed by phagocytic cells. Western blots provide additional evidence for this escape mechanism that appears to occur as a consequence of CD20 loss. Treatment paradigms to prevent this escape, such as use of engineered or alternative anti-CD20 mAbs, may allow for more effective immunotherapy of chronic lymphocytic leukemia.

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Engineered Antibodies with Increased Activity to Recruit Complement

Cited by 266 publications

References 23 publications

The Crystal Structure of the Globular Head of Complement Protein C1q Provides a Basis for Its Versatile Recognition Properties

The Crystal Structure of the Globular Head of Complement Protein C1q Provides a Basis for Its Versatile Recognition Properties

Modulation of the Effector Functions of a Human IgG1 through Engineering of Its Hinge Region

Rituximab Infusion Promotes Rapid Complement Depletion and Acute CD20 Loss in Chronic Lymphocytic Leukemia

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