Use of human germline genes in a CDR homology-based approach to antibody humanization

We report a new method of humanizing antibodies by complementarity determining region (CDR) grafting. Our method differs from others in that we choose human framework sequences from the set of human germline genes based on the structural similarity of the human CDRs to those of the mouse antibody to be humanized. The structural similarity is evaluated by scoring residue-to-residue homology of the mouse CDRs to human candidates with the same Chothia canonical structures. The method is illustrated with the humanization of the anti-lysozyme antibody D1.3.     

Generation and characterization of a recombinant human CCR5-specific antibody. A phage display approach for rabbit antibody humanization

We describe the isolation of a CCR5-specific antibody, ST6, from an antibody phage display library generated from an immune rabbit. ST6 was previously shown to efficiently prevent the surface expression of CCR5 when expressed intracellularly (Steinberger, P., Andris-Widhopf, J., Buhler, B., Torbett, B. E., and Barbas, C. F., III (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 805-810). Because ST6 has therapeutic potential in human immunodeficiency virus, type 1 disease, its humanization was desired to minimize the potential for immunogenicity. ST6 was humanized using a phage display-based approach. Like the parental rabbit clone, the humanized version ST6/34 efficiently prevented the surface expression of CCR5. The conserved linear peptide epitope bound by these antibodies was mapped using phage display. Both ST6 as well as the humanized anti-CCR5 antibody ST6/34 were produced as complete IgG antibodies and shown to bind to cell surface CCR5.     

Structural insights into humanization of anti-tissue factor antibody 10H10

Murine antibody 10H10 raised against human tissue factor is unique in that it blocks the signaling pathway, and thus inhibits angiogenesis and tumor growth without interfering with coagulation. As a potential therapeutic, the antibody was humanized in a two-step procedure. Antigen-binding loops were grafted onto selected human frameworks and the resulting chimeric antibody was subjected to affinity maturation by using phage display libraries. The results of humanization were analyzed from the structural perspective through comparison of the structure of a humanized variant with the parental mouse antibody. This analysis revealed several hot spots in the framework region that appear to affect antigen binding, and therefore should be considered in human germline selection. In addition, some positions in the Vernier zone, e.g., residue 71 in the heavy chain, that are traditionally thought to be crucial appear to tolerate amino acid substitutions without any effect on binding. Several humanized variants were produced using both short and long forms of complementarity-determining region (CDR) H2 following the difference in the Kabat and Martin definitions. Comparison of such pairs indicated consistently higher thermostability of the variants with short CDR H2. Analysis of the binding data in relation to the structures singled out the ImMunoGeneTics information system® germline IGHV1-2*01 as dubious owing to two potentially destabilizing mutations as compared to the other alleles of the same germline and to other human germlines.     

Single cycle structure-based humanization of an anti-nerve growth factor therapeutic antibody

Most forms of chronic pain are inadequately treated by present therapeutic options. Compelling evidence has accumulated, demonstrating that Nerve Growth Factor (NGF) is a key modulator of inflammatory and nociceptive responses, and is a promising target for the treatment of human pathologies linked to chronic and inflammatory pain. There is therefore a growing interest in the development of therapeutic molecules antagonising the NGF pathway and its nociceptor sensitization actions, among which function-blocking anti-NGF antibodies are particularly relevant candidates.In this respect, the rat anti-NGF αD11 monoclonal antibody (mAb) is a potent antagonist, able to effectively antagonize rodent and human NGF in a variety of in vitro and in vivo systems. Here we show that mAb αD11 displays a significant analgesic effect in two different models of persistent pain in mice, with a remarkable long-lasting activity. In order to advance αD11 mAb towards its clinical application in man, anti-NGF αD11 mAb was humanized by applying a novel single cycle strategy based on the a priori experimental determination of the crystal and molecular structure of the parental Fragment antigen-binding (Fab). The humanized antibody (hum-αD11) was tested in vitro and in vivo, showing that the binding mode and the NGF neutralizing biological activities of the parental antibody are fully preserved, with even a significant affinity improvement. The results firmly establish hum-αD11 as a lead candidate for clinical applications in a therapeutic area with a severe unmet medical need. More generally, the single-cycle structure-based humanization method represents a considerable improvement over the standard humanization methods, which are intrinsically empirical and require several refinement cycles.     

VH-VL orientation prediction for antibody humanization candidate selection: A case study

Antibody humanization describes the procedure of grafting a non-human antibody's complementarity-determining regions, i.e., the variable loop regions that mediate specific interactions with the antigen, onto a β-sheet framework that is representative of the human variable region germline repertoire, thus reducing the number of potentially antigenic epitopes that might trigger an anti-antibody response. The selection criterion for the so-called acceptor frameworks (one for the heavy and one for the light chain variable region) is traditionally based on sequence similarity. Here, we propose a novel approach that selects acceptor frameworks such that the relative orientation of the 2 variable domains in 3D space, and thereby the geometry of the antigen-binding site, is conserved throughout the process of humanization. The methodology relies on a machine learning-based predictor of antibody variable domain orientation that has recently been shown to improve the quality of antibody homology models. Using data from 3 humanization campaigns, we demonstrate that preselecting humanization variants based on the predicted difference in variable domain orientation with regard to the original antibody leads to subsets of variants with a significant improvement in binding affinity.     

Critical contribution of VH-VL interaction to reshaping of an antibody: the case of humanization of anti-lysozyme antibody, HyHEL-10

To clarify the effects of humanizing a murine antibody on its specificity and affinity for its target, we examined the interaction between hen egg white lysozyme (HEL) and its antibody, HyHEL-10 variable domain fragment (Fv). We selected a human antibody framework sequence with high homology, grafted sequences of six complementarity-determining regions of murine HyHEL-10 onto the framework, and investigated the interactions between the mutant Fvs and HEL. Isothermal titration calorimetry indicated that the humanization led to 10-fold reduced affinity of the antibody for its target, due to an unfavorable entropy change. Two mutations together into the interface of the variable domains, however, led to complete recovery of antibody affinity and specificity for the target, due to reduction of the unfavorable entropy change. X-ray crystallography of the complex of humanized antibodies, including two mutants, with HEL demonstrated that the complexes had almost identical structures and also paratope and epitope residues were almost conserved, except for complementary association of variable domains. We conclude that adjustment of the interfacial structures of variable domains can contribute to the reversal of losses of affinity or specificity caused by humanization of murine antibodies, suggesting that appropriate association of variable domains is critical for humanization of murine antibodies without loss of function.     

A general approach to antibody thermostabilization

Antibody engineering to enhance thermostability may enable further application and ease of use of antibodies across a number of different areas. A modified human IgG framework has been developed through a combination of engineering approaches, which can be used to stabilize antibodies of diverse specificity. This is achieved through a combination of complementarity-determining region (CDR)-grafting onto the stable framework, mammalian cell display and in vitro somatic hypermutation (SHM). This approach allows both stabilization and maturation to affinities beyond those of the original antibody, as shown by the stabilization of an anti-HA33 antibody by approximately 10°C and affinity maturation of approximately 300-fold over the original antibody. Specificities of 10 antibodies of diverse origin were successfully transferred to the stable framework through CDR-grafting, with 8 of these successfully stabilized, including the therapeutic antibodies adalimumab, stabilized by 9.9°C, denosumab, stabilized by 7°C, cetuximab stabilized by 6.9°C and to a lesser extent trastuzumab stabilized by 0.8°C. This data suggests that this approach may be broadly useful for improving the biophysical characteristics of antibodies across a number of applications.     

A general approach to antibody thermostabilization

Antibody engineering to enhance thermostability may enable further application and ease of use of antibodies across a number of different areas. A modified human IgG framework has been developed through a combination of engineering approaches, which can be used to stabilize antibodies of diverse specificity. This is achieved through a combination of complementarity-determining region (CDR)-grafting onto the stable framework, mammalian cell display and in vitro somatic hypermutation (SHM). This approach allows both stabilization and maturation to affinities beyond those of the original antibody, as shown by the stabilization of an anti-HA33 antibody by approximately 10°C and affinity maturation of approximately 300-fold over the original antibody. Specificities of 10 antibodies of diverse origin were successfully transferred to the stable framework through CDR-grafting, with 8 of these successfully stabilized, including the therapeutic antibodies adalimumab, stabilized by 9.9°C, denosumab, stabilized by 7°C, cetuximab stabilized by 6.9°C and to a lesser extent trastuzumab stabilized by 0.8°C. This data suggests that this approach may be broadly useful for improving the biophysical characteristics of antibodies across a number of applications.     

Use of a peptide mimotope to guide the humanization of MRK-16, an anti-P-glycoprotein monoclonal antibody.

A mimotope-guided strategy for engineering antibodies directed against orphan targets or antigens that are difficult to purify was developed and used to humanize the murine MRK-16 monoclonal antibody (mAb). MRK-16 recognizes a conformational epitope of a 170-kDa membrane protein, termed P-glycoprotein (P-gp). Elevated expression of P-gp on tumor cells is associated with resistance to cytotoxic drugs, a major obstacle in chemotherapy. Murine MRK-16 was used to enrich and screen a phage-displayed peptide library to identify reactive mimotopes. One peptide, termed ALR1, was enriched to a greater extent than others and subsequently was expressed as a fusion protein with glutathione S-transferase. ALR1 fusion protein bound MRK-16 specifically and inhibited binding of MRK-16 to cells expressing elevated levels of P-gp. To humanize MRK-16, the murine complementarity determining regions were grafted onto homologous human heavy and light chain variable region frameworks. Framework residues that differed between the murine MRK-16 and the homologous human templates were analyzed and subsequently, five framework positions potentially important for maintaining the specificity and affinity of MRK-16 were identified. A combinatorial library consisting of 32 variants encoding all possible combinations of murine and human residues at the five differing framework positions was expressed in a phage system. In the absence of purified P-gp, ALR1 fusion protein was used as surrogate antigen to screen the antibody library to identify the framework combination that most preserved the binding activity of the mAb. On the basis of the initial screening against the mimotope four antibody variants were selected for further characterization. The binding affinity of these variants for the ALR1 fusion protein correlated with their binding to cells expressing elevated levels of P-gp. Thus, peptide mimotopes which can be identified for virtually any antibody including those that recognize conformational or carbohydrate epitopes, can serve as antigen templates for antibody engineering.     

Beyond CDR-grafting: Structure-guided humanization of framework and CDR regions of an anti-myostatin antibody

Antibodies are an important class of biotherapeutics that offer specificity to their antigen, long half-life, effector function interaction and good manufacturability. The immunogenicity of non-human-derived antibodies, which can be a major limitation to development, has been partially overcome by humanization through complementarity-determining region (CDR) grafting onto human acceptor frameworks. The retention of foreign content in the CDR regions, however, is still a potential immunogenic liability. Here, we describe the humanization of an anti-myostatin antibody utilizing a 2-step process of traditional CDR-grafting onto a human acceptor framework, followed by a structure-guided approach to further reduce the murine content of CDR-grafted antibodies. To accomplish this, we solved the co-crystal structures of myostatin with the chimeric (Protein Databank (PDB) id 5F3B) and CDR-grafted anti-myostatin antibody (PDB id 5F3H), allowing us to computationally predict the structurally important CDR residues as well as those making significant contacts with the antigen. Structure-based rational design enabled further germlining of the CDR-grafted antibody, reducing the murine content of the antibody without affecting antigen binding. The overall “humanness” was increased for both the light and heavy chain variable regions.     

Construction of a humanized antibody to hepatitis B surface antigen by specificity-determining residues (SDR)-grafting and de-immunization

We previously constructed a humanized antibody, HuS10, by grafting the complementarity-determining regions (CDRs) of a parental murine monoclonal antibody into the homologous human antibody sequences. This process is termed CDR grafting. Some residues that were thought to affect the CDR loops and stabilize the structure of the variable regions were retained in the framework region. HuS10 exhibited in vivo virus-neutralizing activity, but its murine content had the potential to elicit immune responses in patients. In this study, to minimize the immunogenic potential of HuS10, we replaced 17 mouse residues in HuS10 with the comparable human residues using specificity-determining residue (SDR)-grafting and de-immunization methods. The resultant humanized antibody, HzS-III, had the same affinity and epitope specificity as HuS10 and had reduced immunogenic potential, as assessed by T-cell epitope analysis. Thus, SDR grafting in combination with de-immunization may be a useful strategy for minimizing the immunogenicity of humanized antibodies. In addition, HzS-III may be a good candidate for immunoprophylaxis of HBV infection.     

Synthetic approach to the generation of antibody diversity

The in vitro antibody discovery technologies revolutionized the generation of target-specific antibodies that traditionally relied on the humoral response of immunized animals. An antibody library, a large collection of diverse, pre-constructed antibodies, can be rapidly screened using in vitro display technologies such as phage display. One of the keys to successful in vitro antibody discovery is the quality of the library diversity. Antibody diversity can be obtained either from natural B-cell sources or by the synthetic methods that combinatorially generate random nucleotide sequences. While the functionality of a natural antibody library depends largely upon the library size, various other factors can affect the quality of a synthetic antibody library, making the design and construction of synthetic antibody libraries complicated and challenging. In this review, we present various library designs and diversification methods for synthetic antibody library. From simple degenerate oligonucleotide synthesis to trinucleotide synthesis to physicochemically optimized library design, the synthetic approach is evolving beyond the simple emulation of natural antibodies, into a highly sophisticated method that is capable of producing high quality antibodies suitable for therapeutic, diagnostic, and other demanding applications. [BMB Reports 2015; 48(9): 489-494]     

A new approach to microhomogenization

A versatile microhomogenizer is described which is capable of handling sample volumes ranging from 1 μl to several millititers. The sample and homogenization buffer are sealed within a segment of flexible plastic tubing. The sample is disrupted by rubbing or rolling a metal rod back and forth over the length of tubing. Centrifugation of the homogenate is performed in the same sealed tubing segment. This method can be used to homogenize single cells.     

Non-human primate immune libraries combined with germline humanization: An (almost) new,and powerful approach for the isolation of therapeutic antibodies

Panning of libraries constructed from immunised non-human primates (NHP) has not been widely used, even though this has proven to be a successful approach for the isolation of human-like antibody fragments with affinities in the nanomolar to the picomolar range. As recently demonstrated, after initial isolation of antibodies with such high affinities, germline humanization may be applied to these Fabs or scFvs to increase the similarity of their framework regions with those encoded by human germline genes. ‘Germlinized’ antibody fragments may be converted to full size IgGs; indications are given that these IgGs could be better tolerated in clinical use than human antibodies. The use of the combination of NHP immune libraries and germline humanization thus may compete with use of libraries of human origin, whether naïve or immune, and with synthetic libraries. In this report, the various approaches will be compared, and advantages of the two-step NHP-based method, as well as corresponding intellectual property aspects, will be discussed.Key words: non-human primate, antibody, naïve library, synthetic library, immune library, affinity, epitope, tolerance, patent     

Labelled antibody membrane assay for parathyroid hormone a new approach to the measurement of receptor bound hormone.

A new method is described for the measurement of hormone bound to membrane receptors. Antibodies specific for the C-terminal and N-terminal regions of parathyroid hormone were labelled with ¹²⁵I and incubated with renal membranes which had been previously incubated with unlabelled hormone. The uptake of hormone demonstrated pH and time dependence and was a saturable process. Treatment of the membranes with acid or heating to 100°C, or inactivation of the hormone with hydrogen peroxide, completely abolished detectable hormone uptake to the membranes.     

Highly specific off-target binding identified and eliminated during the humanization of an antibody against FGF receptor 4

Off-target binding can significantly affect the pharmacokinetics (PK), tissue distribution, efficacy and toxicity of a therapeutic antibody. Herein we describe the development of a humanized anti- fibroblast growth factor receptor 4 (FGFR4) antibody as a potential therapeutic for hepatocellular carcinoma (HCC). A chimeric anti FGFR4 monoclonal antibody (chLD1) was previously shown to block ligand binding and to inhibit FGFR4 mediated signaling as well as tumor growth in vivo. A humanized version of chLD1, hLD1.vB, had similar binding affinity and in vitro blocking activity, but it exhibited rapid clearance, poor target tissue biodistribution and limited efficacy when compared to chLD1 in a HUH7 human HCC xenograft mouse model. These problems were traced to instability of the molecule in rodent serum. Size exclusion high performance liquid chromatography, immunoprecipitation and mass spectral sequencing identified a specific interaction between hLD1.vB and mouse complement component 3 (C3). A PK study in C3 knock-out mice further confirmed this specific interaction. Subsequently, an affinity-matured variant derived from hLD1.vB (hLD1.v22), specifically selected for its lack of binding to mouse C3 was demonstrated to have a PK profile and in vivo efficacy similar to that of chLD1 in mice. Although reports of non-specific off-target binding have been observed for other antibodies, this represents the first report identifying a specific off-target interaction that affected disposition and biological activity. Screens developed to identify general non-specific interactions are likely to miss the rare and highly specific cross-reactivity identified in this study, thus highlighting the importance of animal models as a proxy for avoiding unexpected clinical outcomes.