Construction and characterization of a high-affinity humanized SM5-1 monoclonal antibody

SM5-1 is a mouse monoclonal antibody which has a high specificity for melanoma, hepatocellular carcinoma, and breast cancer, making it a promising candidate for cancer targeting therapy. We have therefore attempted to construct a humanized antibody of SM5-1 to minimize its immunogenicity for potential clinical use. Using a molecular model of SM5-1 built by computer-assisted homology modeling, framework region (FR) residues of potential importance to the antigen binding were identified. Then, a humanized version of SM5-1 was generated by transferring these mouse key FR residues onto a human framework that was selected based on homology to the mouse framework, together with the mouse complementarity-determining region (CDR) residues. This humanized antibody retained only six murine residues outside of the CDRs but was shown to possess affinity and specificity comparable to that of the parental antibody, suggesting that it might have the potential to be developed for future clinical use.     

Humanization of a murine monoclonal antibody by simultaneous optimization of framework and CDR residues.

Optimal protein function often depends on co-operative interactions between amino acid residues distant in the protein primary sequence yet spatially near one another following protein folding. For example, antibody affinity is influenced by interactions of framework residues with complementarity-determining region (CDR) residues. However, despite the abundance of antibody structural information and computational tools the humanization of rodent antibodies for clinical use often results in a significant loss of affinity. To date, antibody engineering efforts have focused either on optimizing CDR residues involved in antigen binding or on optimizing antibody framework residues that serve critical roles in preserving the conformation of CDRs. In the present study a new approach which permits the rapid identification of co-operatively interacting framework and CDR residues was used to simultaneously humanize and optimize a murine antibody directed against CD40. Specifically, a combinatorial library that examined eight potentially important framework positions concomitantly with focused CDR libraries consisting of variants containing random single amino acid mutations in the third CDR of the heavy and light chains was expressed. Multiple anti-CD40 Fab variants containing as few as one murine framework residue and displaying up to approximately 500-fold higher affinity than the initial chimeric Fab were identified. The higher affinity humanized variants demonstrated a co-operative interaction between light chain framework residue Y49 and heavy chain CDR3 residue R/K101 (coupling energy, DeltaGI=0.9 kcal/mol). Screening of combinatorial framework-CDR libraries permits identification of monoclonal antibodies (mAb) with structures optimized for function, including instances in which the antigen induces conformational changes in the mAb. Moreover, the enhanced humanized variants contain fewer murine framework residues and could not be identified by sequential in vitro humanization and affinity muturation strategies. This approach to identifying co-operatively interacting residues is not restricted to antibody-antigen interactions and consequently, may be used broadly to gain insight into protein structure-function relationships, including proteins that serve as catalysts.     

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.     

Humanization of anti-human insulin receptor antibody for drug targeting across the human blood-brain barrier

A murine monoclonal antibody (MAb) to the human insulin receptor (HIR) has been engineered for use as a brain drug delivery system for transport across the human blood-brain barrier (BBB). The HIRMAb was humanized by complementarity determining region (CDR) grafting on the framework regions (FR) of the human B43 IgG heavy chain and the human REI kappa light chain. A problem encountered in the humanization process was the poor secretion of the CDR-grafted HIRMAb by myeloma cells. This problem was solved by the production of human/mouse hybrids of the engineered heavy chain variable region (VH), which led to the replacement of five amino acids in the FR3 of the VH with original murine amino acids. No replacement of FR amino acids in the light chain variable region (VL) was required. The affinity of the humanized HIRMAb for the HIR was decreased 27% relative to the murine HIRMAb. The humanized HIRMAb avidly bound to the HIR of isolated human brain capillaries, which are used as an in vitro model system of the human BBB. The HIRMAb cross reacts with the HIR of Old World primates such as the Rhesus monkey. The humanized HIRMAb was radiolabeled with 125-iodine, and injected intravenously into an adult, anesthetized Rhesus monkey. Brain scanning showed the humanized HIRMAb was rapidly transported into all parts of the primate brain after intravenous administration. The humanized HIRMAb may be used as a brain drug and gene delivery system for the targeting of large molecule therapeutics across the BBB in humans.     

Chimeric and humanized antibodies with specificity for the CD33 antigen.

L and H chain cDNAs of M195, a murine mAb that binds to the CD33 Ag on normal and leukemic myeloid cells, were cloned. The cDNAs were used in the construction of mouse/human IgG1 and IgG3 chimeric antibodies. In addition, humanized antibodies were constructed which combined the complementarity-determining regions of the M195 antibody with human framework and constant regions. The human framework was chosen to maximize homology with the M195 V domain sequence. Moreover, a computer model of M195 was used to identify several framework amino acids that are likely to interact with the complementarity-determining regions, and these residues were also retained in the humanized antibodies. Unexpectedly, the humanized IgG1 and IgG3 M195 antibodies, which have reshaped V regions, have higher apparent binding affinity for the CD33 Ag than the chimeric or mouse antibodies.     

Construction, affinity maturation, and biological characterization of an anti-tumor-associated glycoprotein-72 humanized antibody

The tumor-associated glycoprotein (TAG)-72 is expressed in the majority of human adenocarcinomas but is rarely expressed in most normal tissues, which makes it a potential target for the diagnosis and therapy of a variety of human cancers. Here we describe the construction, affinity maturation, and biological characterization of an anti-TAG-72 humanized antibody with minimum potential immunogenicity. The humanized antibody was constructed by grafting only the specificity-determining residues (SDRs) within the complementarity-determining regions (CDRs) onto homologous human immunoglobulin germ line segments while retaining two mouse heavy chain framework residues that support the conformation of the CDRs. The resulting humanized antibody (AKA) showed only about 2-fold lower affinity compared with the original murine monoclonal antibody CC49 and 27-fold lower reactivity to patient serum compared with the humanized antibody HuCC49 that was constructed by CDR grafting. The affinity of AKA was improved by random mutagenesis of the heavy chain CDR3 (HCDR3). The highest affinity variant (3E8) showed 22-fold higher affinity compared with AKA and retained the original epitope specificity. Mutational analysis of the HCDR3 residues revealed that the replacement of Asn(97) by isoleucine or valine was critical for the affinity maturation. The 3E8 labeled with (125)I or (131)I showed efficient tumor targeting or therapeutic effects, respectively, in athymic mice with human colon carcinoma xenografts, suggesting that 3E8 may be beneficial for the diagnosis and therapy of tumors expressing TAG-72.     

Guided selection of human antibody light chains against TAG-72 using a phage display chain shuffling approach

To enhance therapeutic potential of murine monoclonal antibody, humanization by CDR grafting is usually used to reduce immunogenic mouse residues. Most humanized antibodies still have mouse residues critical for antigen binding, but the mouse residues may evoke immune responses in humans. Previously, we constructed a new humanized version (AKA) of mouse CC49 antibody specific for tumor-associated glycoprotein, TAG-72. In this study, to select a completely human antibody light chain against TAG-72, guided selection strategy using phage display was used. The heavy chain variable region (VH) of AKA was used to guide the selection of a human TAG-72-specific light chain variable region (VL) from a human VL repertoire constructed from human PBL. Most of the selected VLs were identified to be originated from the members of the human germline VK1 family, whereas the VL of AKA is more homologous to the VK4 family. Competition binding assay of the selected Fabs with mouse CC49 suggested that the epitopes of the Fabs overlap with that of CC49. In addition, they showed better antigen-binding affinity compared to parental AKA. The selected human VLs may be used to guide the selection of human VHs to get completely human anti-TAG72 antibody.     

Structure guided homology model based design and engineering of mouse antibodies for humanization

No universal strategy exists for humanizing mouse antibodies, and most approaches are based on primary sequence alignment and grafting. Although this strategy theoretically decreases the immunogenicity of mouse antibodies, it neither addresses conformational changes nor steric clashes that arise due to grafting of human germline frameworks to accommodate mouse CDR regions. To address these issues, we created and tested a structure-based biologic design approach using a de novo homology model to aid in the humanization of 17 unique mouse antibodies. Our approach included building a structure-based de novo homology model from the primary mouse antibody sequence, mutation of the mouse framework residues to the closest human germline sequence and energy minimization by simulated annealing on the humanized homology model. Certain residues displayed force field errors and revealed steric clashes upon closer examination. Therefore, further mutations were introduced to rationally correct these errors. In conclusion, use of de novo antibody homology modeling together with simulated annealing improved the ability to predict conformational and steric clashes that may arise due to conversion of a mouse antibody into the humanized form and would prevent its neutralization when administered in vivo. This design provides a robust path towards the development of a universal strategy for humanization of mouse antibodies using computationally derived antibody homologous structures.     

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.     

3D modeling and characterization of the human CD115 monoclonal antibody H27K15 epitope and design of a chimeric CD115 target

The humanized monoclonal antibody H27K15 specifically targets human CD115, a type III tyrosine kinase receptor involved in multiple cancers and inflammatory diseases. Binding of H27K15 to hCD115 expressing cells inhibits the functional effect of colony-stimulating factor-1 (CSF-1), in a non-competitive manner. Both homology modeling and docking programs were used here to model the human CD115 extracellular domains, the H27K15 variable region and their interaction. The resulting predicted H27K15 epitope includes mainly the D1 domain in the N-terminal extracellular region of CD115 and some residues of the D2 domain. Sequence alignment with the non-binding murine CD115, enzyme-linked immunosorbent assay, nuclear magnetic resonance spectroscopy and affinity measurements by quartz crystal microbalance revealed critical residues of this epitope that are essential for H27K15 binding. A combination of computational simulations and biochemical experiments led to the design of a chimeric CD115 carrying the human epitope of H27K15 in a murine CD115 backbone that is able to bind both H27K15 as well as the murine ligands CSF-1 and IL-34. These results provide new possibilities to minutely study the functional effects of H27K15 in a transgenic mouse that would express this chimeric molecule.     

3D modeling and characterization of the human CD115 monoclonal antibody H27K15 epitope and design of a chimeric CD115 target

The humanized monoclonal antibody H27K15 specifically targets human CD115, a type III tyrosine kinase receptor involved in multiple cancers and inflammatory diseases. Binding of H27K15 to hCD115 expressing cells inhibits the functional effect of colony-stimulating factor-1 (CSF-1), in a non-competitive manner. Both homology modeling and docking programs were used here to model the human CD115 extracellular domains, the H27K15 variable region and their interaction. The resulting predicted H27K15 epitope includes mainly the D1 domain in the N-terminal extracellular region of CD115 and some residues of the D2 domain. Sequence alignment with the non-binding murine CD115, enzyme-linked immunosorbent assay, nuclear magnetic resonance spectroscopy and affinity measurements by quartz crystal microbalance revealed critical residues of this epitope that are essential for H27K15 binding. A combination of computational simulations and biochemical experiments led to the design of a chimeric CD115 carrying the human epitope of H27K15 in a murine CD115 backbone that is able to bind both H27K15 as well as the murine ligands CSF-1 and IL-34. These results provide new possibilities to minutely study the functional effects of H27K15 in a transgenic mouse that would express this chimeric molecule.     

SDR grafting--a new approach to antibody humanization

A major impediment to the clinical utility of the murine monoclonal antibodies is their potential to elicit human anti-murine antibody (HAMA) response in patients. To circumvent this problem, murine antibodies have been genetically manipulated to progressively replace their murine content with the amino acid residues present in their human counterparts. To that end, murine antibodies have been humanized by grafting their complementarity determining regions (CDRs) onto the variable light (V(L)) and variable heavy (V(H)) frameworks of human immunoglobulin molecules, while retaining those murine framework residues deemed essential for the integrity of the antigen-combining site. However, the xenogeneic CDRs of the humanized antibodies may evoke anti-idiotypic (anti-Id) response in patients. To minimize the anti-Id response, a procedure to humanize xenogeneic antibodies has been described that is based on grafting, onto the human frameworks, only the specificity determining residues (SDRs), the CDR residues that are most crucial in the antibody-ligand interaction. The SDRs are identified through the help of the database of the three-dimensional structures of the antigen-antibody complexes of known structures or by mutational analysis of the antibody-combining site. An alternative approach to humanization, which involves retention of more CDR residues, is based on grafting of the 'abbreviated' CDRs, the stretches of CDR residues that include all the SDRs. A procedure to assess the reactivity of the humanized antibody to sera from patients who had been administered the murine antibody has also been described.     

The immunogenicity of humanized and fully human antibodies

Monoclonal antibodies represent an attractive therapeutic tool as they are highly specific for their targets, convey effector functions and enjoy robust manufacturing procedures. Humanization of murine monoclonal antibodies has vastly improved their in vivo tolerability. Humanization, the replacement of mouse constant regions and V framework regions for human sequences, results in a significantly less immunogenic product. However, some humanized and even fully human sequence-derived antibody molecules still carry immunological risk. To more fully understand the immunologic potential of humanized and human antibodies, we analyzed CD4+ helper T cell epitopes in a set of eight humanized antibodies. The antibodies studied represented a number of different VH and VL family members carrying unique CDR regions. In spite of these differences, CD4+ T cell epitopes were found only in CDR-sequence containing regions. We were able to incorporate up to two amino acid modifications in a single epitope that reduced the immunogenic potential while retaining full biologic function. We propose that immunogenicity will always be present in some antibody molecules due to the nature of the antigen-specific combining sites. A consequence of this result is modifications to reduce immunogenicity will be centered on the affinity-determining regions. Modifications to CDR regions can be designed that reduce the immunogenic potential while maintaining the bioactivity of the antibody molecule.     

Stability of murine, chimeric and humanized antibodies against pre-S2 surface antigen of hepatitis B virus

We have constructed a humanized antibody with specificity for the pre-S2 surface antigen of hepatitis B virus (HBV) by grafting the complementarity determining regions (CDRs) of parental murine monoclonal antibody (mAb) into human anti-Sm antibody framework regions. The humanized antibody has a substitution at position 94 in a framework region of the heavy chain variable region, and exhibits the same antigen binding affinity as the parental murine monoclonal and chimeric antibodies. In order to assess the stability of these antibodies, thermal inactivation of the parental, chimeric and humanized antibodies was analyzed. Fifty percent inactivation of the chimeric and humanized antibodies was observed at 63.7 degrees C and 68.7 degrees C, respectively, compared to 55.0 degrees C for murine antibody. The humanized antibody also exhibited increased stability against denaturant. Guanidine-induced unfolding monitored by the changes in fluorescence intensity at 360 nm showed that midpoints of the transition of the chimeric and humanized antibodies were 2.47 M and 2.56 M, respectively, whereas that of the murine antibody was 1.36 M.     

Light-chain framework region residue Tyr71 of chimeric B72.3 antibody plays an important role in influencing the TAG72 antigen binding.

The crystallographic study of chimeric B72.3 antibody illustrated that there are three FR side-chain interactions with either CDR residue's side chain or main chain. For example, hydrogen bonds are formed between the hydroxyl group of threonine at L5 in FR1 and the guanidinal nitrogen group of arginine at L24 in CDR1, between the hydroxyl group of tyrosine at L36 in FR2 and the amide nitrogen group of glutamine at L89 in CDR3 and between the hydroxyl group of tyrosine at L71 in FR3 and the carbonyl group of isoleucine at L29 as well as the amide nitrogen group of serine at L31 in CDR1. Elimination of these hydrogen bonds at these FR positions may affect CDR loop conformations. To confirm these assumptions, we altered these FR residues by site-directed mutagenesis and determined binding affinities of these mutant chimeric antibodies for the TAG72 antigen. We found that the substitution of tyrosine by phenylalanine at L71, altering main-chain hydrogen bonds, significantly reduced the binding affinity for the TAG72 antigen by 23-fold, whereas the substitution of threonine and tyrosine by alanine and phenylalanine at L5 and L36, eliminating hydrogen bonds to side-chain atoms, did not affect the binding affinity for the TAG72 antigen. Our results indicate that the light-chain FR residue tyrosine at L71 of chimeric B72.3 antibody plays an important role in influencing the TAG72 antigen binding. Our results will thus be of importance when the humanized B72.3 antibody is constructed, since this important mouse FR residue tyrosine at L71 must be maintained.     

The immunogenicity of humanized and fully human antibodies: Residual immunogenicity resides in the CDR regions

Monoclonal antibodies represent an attractive therapeutic tool as they are highly specific for their targets, convey effector functions and enjoy robust manufacturing procedures. Humanization of murine monoclonal antibodies has vastly improved their in vivo tolerability. Humanization, the replacement of mouse constant regions and V framework regions for human sequences, results in a significantly less immunogenic product. However, some humanized and even fully human sequence-derived antibody molecules still carry immunological risk. to more fully understand the immunologic potential of humanized and human antibodies, we analyzed CD4⁺ helper T cell epitopes in a set of eight humanized antibodies. the antibodies studied represented a number of different VH and VL family members carrying unique CDR regions. In spite of these differences, CD4⁺ T cell epitopes were found only in CDR-sequence containing regions. We were able to incorporate up to two amino acid modifications in a single epitope that reduced the immunogenic potential while retaining full biologic function. We propose that immunogenicity will always be present in some antibody molecules due to the nature of the antigen-specific combining sites. A consequence of this result is modifications to reduce immunogenicity will be centered on the affinity-determining regions. Modifications to CDR regions can be designed that reduce the immunogenic potential while maintaining the bioactivity of the antibody molecule.Key words: therapeutic, antibody, immunogenicity, deimmunizing, epitope     

Construction and characterization of functional anti-epiregulin humanized monoclonal antibodies

Growth factors are implicated in several processes essential for cancer progression. Specifically, epidermal growth factor (EGF) family members, including epiregulin (EREG), are important prognostic factors in many epithelial cancers, and treatments targeting these molecules have recently become available. Here, we constructed and expressed humanized anti-EREG antibodies by variable domain resurfacing based on the three-dimensional (3D) structure of the Fv fragment. However, the initial humanized antibody (HM0) had significantly decreased antigen-binding affinity. Molecular modeling results suggested that framework region (FR) residues latently important to antigen binding included residue 49 of the light chain variable region (VL). Back mutation of the VL49 residue (tyrosine to histidine) generated the humanized version HM1, which completely restored the binding affinity of its murine counterpart. Importantly, only one mutation in the framework may be necessary to recover the binding capability of a humanized antibody. Our data support that HM1 exerts potent antibody-dependent cellular cytotoxicity (ADCC). Hence, this antibody may have potential for further development as a candidate therapeutic agent and research tool.     

Anti-peptide antibodies detect a lupus-related interspecies idiotype that maps to H chain CDR2.

Antibodies to the small nucleoprotein Sm occur spontaneously in human and murine systemic lupus erythematosus. Human and mouse monoclonal anti-Sm autoantibodies designated 4B4 and Y2 share an idiotype (Id) determinant located on the Ig H chain. To understand the molecular basis of this cross-reactivity, the VH regions of both antibodies were sequenced and analyzed for homology. The antibodies showed only 49.6% homology. The second complementary determining region (CDR2) was the most likely candidate for the Id site. To investigate this possibility, rabbit antiserum was made against a peptide corresponding to the CDR2 of 4B4. This antiserum was specific for the immunizing peptide and reacted weakly to a peptide corresponding to the CDR2 of Y2. Anti-CDR2 antibody bound to 4B4 and Y2 but not to other human and mouse mAb. Binding was directed at the H chain when analyzed by Western blots. Anti-CDR2 antibody blocked anti-Id antibody binding to 4B4 and Y2 by 58% and 24%, respectively. These studies suggest that this interspecies Id maps to the H chain CDR2 and that a conserved Id can occur within molecules that are otherwise radically different.     

Humanization and characterization of an anti-human TNF-α murine monoclonal antibody

A murine monoclonal antibody, m357, showing the highly neutralizing activities for human tumor necrosis factor (TNF-α) was chosen to be humanized by a variable domain resurfacing approach. The non-conserved surface residues in the framework regions of both the heavy and light chain variable regions were identified via a molecular modeling of m357 built by computer-assisted homology modeling. By replacing these critical surface residues with the human counterparts, a humanized version, h357, was generated. The humanized h357 IgG(1) was then stably expressed in a mammalian cell line and the purified antibody maintained the high antigen binding affinity as compared with the parental m357 based on a soluble TNF-α neutralization bioassay. Furthermore, h357 IgG(1) possesses the ability to mediate antibody-dependent cell-mediated cytotoxicity and complement dependent cytotoxicity upon binding to cells bearing the transmembrane form of TNF-α. In a mouse model of collagen antibody-induced arthritis, h357 IgG significantly inhibited disease progression by intra-peritoneal injection of 50 μg/mouse once-daily for 9 consecutive days. These results provided a basis for the development of h357 IgG as therapeutic use.     

Evidence for involvement of a hydrophobic patch in framework region 1 of human V4-34-encoded Igs in recognition of the red blood cell I antigen

The monoclonal IgM cold agglutinins that bind to the I/i carbohydrate Ags on the surface of RBCs all have Ig H chains encoded by the V4-34 gene segment. This mandatory use indicates that distinctive amino acid sequences may be involved in recognition. Critical amino acids exist in framework region 1 (FR1) of V4-34-encoded Ig, and these generate a specific Id determinant which apparently lies close to the I binding site. However, I binding by Id-expressing Ig can be modulated by sequences in complementarity-determining region (CDR)(H)3. Examination of the crystal structure of an anti-I cold agglutinin has revealed a hydrophobic patch in FR1 involving residue W7 on beta-strand A and the AVY motif (residues 23-25) on beta-strand B. In this study we used mutagenesis to show that each of the strand components of the hydrophobic patch is required for binding the I carbohydrate Ag. In addition, the crystal structure reveals that amino acids in the carboxyl-terminal region of CDR(H)3 form a surface region adjacent to the hydrophobic patch. We propose that the I carbohydrate Ag interacts simultaneously with the entire hydrophobic patch in FR1 and with the outside surface of CDR(H)3. This interaction could leave most of the conventional binding site available for binding other Ags.