Production and characterization of a humanized single-chain antibody against human integrin alphav beta3 protein

Anti-angiogenesis therapy is an emerging strategy for cancer treatment. This therapy has many advantages over existing treatments, such as fewer side effects, fewer resistance problems, and a broader tumor type spectrum. Integrin αvβ3 is a heterodimeric transmembrane glycoprotein that has been demonstrated to play a key role in tumor angiogenesis and metastasis. We have used a phage antibody display to humanize a mouse monoclonal antibody (mAb E10) against human integrin αvβ3 with a predetermined CDR3 gene. Three human phage antibodies were developed. Analysis of the humanized phage antibodies by phage ELISA revealed that the antibodies retained high antigen-binding activity and detected the same epitope as the parent mAb E10. A humanized single chain Fv (scFv) antibody was expressed in Escherichia coli in a soluble form. Analysis of the purified scFv indicated that it has the same specificity and affinity as the original mAb. Cell viability assays and xenograft model results suggested that the humanized scFv possesses anti-tumor growth activity in vitro and in vivo. This successful production of a humanized scFv with the ability to inhibit αvβ3-mediated cancer cell growth may provide a novel candidate for integrin αvβ3-targeted therapy.     

Defucosylated anti-CCR4 monoclonal antibody exercises potent ADCC-mediated antitumor effect in the novel tumor-bearing humanized NOD/Shi-scid, IL-2Rγnull mouse model

Purpose  There are no suitable small animal models to evaluate human antibody-dependent cellular cytotoxicity (ADCC) in vivo, due to species incompatibilities. Thus, the first aim of this study was to establish a human tumor-bearing mouse model in which human immune cells can engraft and mediate ADCC, but where the endogenous mouse immune cells cannot mediate ADCC. The second aim was to evaluate ADCC mediated in these humanized mice by the defucosylated anti-CC chemokine receptor 4 (CCR4) monoclonal antibody (mAb) which we have developed and which is now in phase I clinical trials. Experimental design  NOD/Shi-scid, IL-2Rγ^null (NOG) mice were the recipients of human immune cells, and CCR4-expressing Hodgkin lymphoma (HL) and cutaneous T-cell lymphoma (CTCL) cell lines were used as target tumors. Results  Humanized mice have been established using NOG mice. The chimeric defucosylated anti-CCR4 mAb KM2760 showed potent antitumor activity mediated by robust ADCC in these humanized mice bearing the HL or CTCL cell lines. KM2760 significantly increased the number of tumor-infiltrating CD56-positive NK cells which mediate ADCC, and reduced the number of tumor-infiltrating FOXP3-positive regulatory T (Treg) cells in HL-bearing humanized mice. Conclusions  Anti-CCR4 mAb could be an ideal treatment modality for many different cancers, not only to directly kill CCR4-expressing tumor cells, but also to overcome the suppressive effect of Treg cells on the host immune response to tumor cells. In addition, using our humanized mice, we can perform the appropriate preclinical evaluation of many types of antibody based immunotherapy.     

Cloning of a hamster anti-mouse CD79B antibody sequences and identification of a new hamster immunoglobulin lambda constant IGLC gene region

Anti-CD79 antibodies have been effective at targeting B cell lymphoma cells and depleting B cells in animal models. In order to engineer recombinant antibodies with additional effector functions in mice, we cloned and sequenced the full-length cDNAs of the heavy and light chain of a hamster anti-mouse CD79B antibody. Although hamster antibodies represent a unique source of monoclonal antibodies against mouse, rat, and human antigens, sequence information of hamster immunoglobulins (IG) is sparse. Here, we report a new hamster (Cricetulus migratorius) IG lambda constant (IGLC) gene region that is most homologous to mouse IGLC2 and IGLC3.     

A complement-dependent cytotoxicity-enhancing anti-CD20 antibody mediating potent antitumor activity in the humanized NOD/Shi-scid, IL-2Rγnull mouse lymphoma model

Engineering the Fc region of monoclonal antibodies (mAb) in order to enhance effector functions such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (CDC) is likely to a be promising approach for next-generation mAb therapy. Here, we report on such an antibody, 113F, a novel CDC-enhancing variant of rituximab, and determine the tumor-associated factors influencing susceptibility to 113F-induced CDC. The latter included the quantity of complement inhibitors present, such as CD55 and CD59. We report that compared to rituximab, 113F mediated highly enhanced CDC against primary CD20-expressing lymphoma cells in vitro. Currently, a major problem in the field of immunotherapy research is the lack of suitable small animal models to evaluate human CDC in vivo. Therefore, we established a novel human tumor-bearing NOD/Shi-scid, IL-2Rγ^null mouse model, in which human complement functions as the CDC mediator. We demonstrated that rituximab exerted significant antitumor effects via human CDC in this humanized mouse. The finding of specific localization of human C1q on CD20-expressing tumor cell membranes was consistent with the observation that human CDC indeed contributed to the antitumor effect in this model. Moreover, 113F exerted significantly more potent antitumor effects than rituximab in this in vivo model. The detection of more abundant dense signals from C1q using 113F compared to rituximab was consistent with the concept that this reagent represented a CDC-enhancing mAb. In the near future, the efficacy of this type of CDC-enhancing antibody will be determined in clinical trials in humans.     

Development of humanized antibodies as cancer therapeutics

Recent success in the development of monoclonal antibody-based anti-cancer drugs has largely benefitted from the advancements made in recombinant technologies and cell culture production. These reagents, derived from the antibodies of mouse origin, while maintaining the exquisite specificity and affinity to the tumor antigens, have low immunogenicity and toxicity in human. High-level expressing cell clones are generated and used to produce large quantities of the recombinant antibodies in bioreactors in order to meet the clinical demand for therapeutic applications. In this report, the systems and general methodologies developed by us to construct and produce humanized antibodies from the parent mouse antibodies are described. Once the humanized antibodies are available, they can be applied in three principal forms for cancer therapy: (1) naked antibodies, (2) drug- or toxin conjugates, and (3) radioconjugates. Using the humanized anti-CD22 (epratuzumab) and anti-carcinoembryonic antigen (ant-CEA; labetuzumab) antibody prototypes, clinical applications of naked and radiolabeled humanized monoclonal antibodies are described.     

Human T cells targeted with anti-T3 cross-linked to antitumor antibody prevent tumor growth in nude mice

Human peripheral blood T cells were tested for the ability to prevent tumor growth in nude mice when targeted with anti-T3 cross-linked to antitumor antibodies. LS174T human colon adenocarcinoma cells were mixed with human PBL coated either with anti-T3 (Fab) cross-linked to 315F6 (Fab) (an antitumor monoclonal antibody) or with no antibody, and were injected subcutaneously into nude mice. Tumor growth was totally inhibited at effector to target (E:T) ratios of 7.0:1 and 2.1:1, and was partially inhibited at 0.7:1 with antibody-coated PBL, but was not inhibited by uncoated PBL. T cell-mediated protection against tumor growth occurred when an antitumor was physically cross-linked to anti-T3. Neither a mixture of unlinked anti-T3 and antitumor antibodies nor anti-human MHC class I cross-linked to antitumor antibody prevented tumor growth. Whereas in vitro cytotoxicity was mediated exclusively by T8+ cells and was augmented by brief exposure of effector cells to IL 2, tumor neutralization in vivo was mediated by both T4+ and T8+ cells and was not significantly stimulated by prior exposure of the cells to IL 2. We conclude that human T cells, when targeted with appropriate antibody heteroaggregates, can specifically inhibit tumor growth at low E:T ratios, and that cells mediating tumor neutralization in vivo may differ from those mediating cytotoxicity in vitro.     

EV20, a Novel Anti-ErbB-3 Humanized Antibody,Promotes ErbB-3 Down-Regulation and Inhibits Tumor Growth In Vivo

ErbB-3 (HER-3) receptor is involved in tumor progression and resistance to therapy. Development of specific inhibitors impairing the activity of ErbB-3 is an attractive tool for cancer therapeutics. MP-RM-1, a murine monoclonal antibody targeting human ErbB-3, has shown anticancer activity in preclinical models. With the aim to provide novel candidates for clinical use, we have successfully generated a humanized version of MP-RM-1. The humanized antibody, named EV20, abrogates both ligand-dependent and ligand-independent receptor signaling of several tumor cell types, strongly promotes ErbB-3 down-regulation, and efficiently and rapidly internalizes into tumor cells. Furthermore, treatment with EV20 significantly inhibits growth of xenografts originating from prostatic, ovarian, and pancreatic cancers as well as melanoma in nude mice. In conclusion, we provide a novel candidate for ErbB-3-targeted cancer therapy.     

Co-transplantation of human hematopoietic stem cells and human breast cancer cells in NSG mice: A novel approach to generate tumor cell specific human antibodies

Humanized tumor mice (HTM) were generated by the co-transplantation of human hematopoietic stem cells and human breast cancer cells overexpressing HER2 into neonatal NOD-scid IL2Rγ^null (NSG) mice. These mice are characterized by the development of a human immune system in combination with human breast cancer growth. Due to concurrent transplantation into newborn mice, transfer of MHC-mismatched tumor cells resulted in solid coexistence and immune cell activation (CD4⁺ T cells, natural killer cells, and myeloid cells), but without evidence for rejection. Histological staining of the spleen of HTM revealed co-localization of human antigen-presenting cells together with human T and B cells allowing MHC-dependent interaction, and thereby the generation of T cell-dependent antibody production. Here, we investigated the capability of these mice to generate human tumor-specific antibodies and correlated immunoglobulin titers with tumor outgrowth. We found detectable IgM and also IgG amounts in the serum of HTM, which apparently controlled tumor development when IgG serum concentrations were above 10 µg/ml. Western blot analyses revealed that the tumor-specific antibodies generated in HTM did not recognize HER2/neu antigens, but different, possibly relevant antigens for breast cancer therapy. In conclusion, HTM offer a novel approach to generate complete human monoclonal antibodies that do not require further genetic manipulation (e. g., humanization) for a potential application in humans. In addition, efficacy and safety of the generated antibodies can be tested in the same mouse model under human-like conditions. This might be of particular interest for cancer subtypes with no currently available antibody therapy.     

Platelets Promote Tumor Growth and Metastasis via Direct Interaction between Aggrus/Podoplanin and CLEC-2

The platelet aggregation-inducing factor Aggrus, also known as podoplanin, is frequently upregulated in several types of tumors and enhances hematogenous metastasis by interacting with and activating the platelet receptor CLEC-2. Thus, Aggrus–CLEC-2 binding could be a therapeutic molecular mechanism for cancer therapy. We generated a new anti-human Aggrus monoclonal antibody, MS-1, that suppressed Aggrus–CLEC-2 binding, Aggrus-induced platelet aggregation, and Aggrus-mediated tumor metastasis. Interestingly, the MS-1 monoclonal antibody attenuated the growth of Aggrus-positive tumors in vivo. Moreover, the humanized chimeric MS-1 antibody, ChMS-1, also exhibited strong antitumor activity against Aggrus-positive lung squamous cell carcinoma xenografted into NOD-SCID mice compromising antibody-dependent cellular cytotoxic and complement-dependent cytotoxic activities. Because Aggrus knockdown suppressed platelet-induced proliferation in vitro and tumor growth of the lung squamous cell carcinoma in vivo, Aggrus may be involved in not only tumor metastasis but also tumor growth by promoting platelet-tumor interaction, platelet activation, and secretion of platelet-derived factors in vivo. Our results indicate that molecular target drugs inhibiting specific platelet–tumor interactions can be developed as antitumor drugs that suppress both metastasis and proliferation of tumors such as lung squamous cell carcinoma.     

Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus

Neutralization of West Nile virus (WNV) in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Using random mutagenesis and yeast surface display, we defined individual contact residues of 14 newly generated monoclonal antibodies against domain III of the WNV E protein. Monoclonal antibodies that strongly neutralized WNV localized to a surface patch on the lateral face of domain III. Convalescent antibodies from individuals who had recovered from WNV infection also detected this epitope. One monoclonal antibody, E16, neutralized 10 different strains in vitro, and showed therapeutic efficacy in mice, even when administered as a single dose 5 d after infection. A humanized version of E16 was generated that retained antigen specificity, avidity and neutralizing activity. In postexposure therapeutic trials in mice, a single dose of humanized E16 protected mice against WNV-induced mortality, and may therefore be a viable treatment option against WNV infection in humans.     

Phosphodiesterase Type 5 Inhibitors Increase Herceptin Transport and Treatment Efficacy in Mouse Metastatic Brain Tumor Models

Background

Chemotherapeutic drugs and newly developed therapeutic monoclonal antibodies are adequately delivered to most solid and systemic tumors. However, drug delivery into primary brain tumors and metastases is impeded by the blood-brain tumor barrier (BTB), significantly limiting drug use in brain cancer treatment.

Methodology/Principal Findings

We examined the effect of phosphodiesterase 5 (PDE5) inhibitors in nude mice on drug delivery to intracranially implanted human lung and breast tumors as the most common primary tumors forming brain metastases, and studied underlying mechanisms of drug transport. In vitro assays demonstrated that PDE5 inhibitors enhanced the uptake of [¹⁴C]dextran and trastuzumab (Herceptin®, a humanized monoclonal antibody against HER2/neu) by cultured mouse brain endothelial cells (MBEC). The mechanism of drug delivery was examined using inhibitors for caveolae-mediated endocytosis, macropinocytosis and coated pit/clathrin endocytosis. Inhibitor analysis strongly implicated caveolae and macropinocytosis endocytic pathways involvement in the PDE5 inhibitor-enhanced Herceptin uptake by MBEC. Oral administration of PDE5 inhibitor, vardenafil, to mice with HER2-positive intracranial lung tumors led to an increased tumor permeability to high molecular weight [¹⁴C]dextran (2.6-fold increase) and to Herceptin (2-fold increase). Survival time of intracranial lung cancer-bearing mice treated with Herceptin in combination with vardenafil was significantly increased as compared to the untreated, vardenafil- or Herceptin-treated mice (p<0.01). Log-rank survival analysis of mice bearing HER2-positive intracranial breast tumor also showed a significant survival increase (p<0.02) in the group treated with Herceptin plus vardenafil as compared to other groups. However, vardenafil did not exert any beneficial effect on survival of mice bearing intracranial breast tumor with low HER2 expression and co-treated with Herceptin (p>0.05).

Conclusions/Significance

These findings suggest that PDE5 inhibitors may effectively modulate BTB permeability, and enhance delivery and therapeutic efficacy of monoclonal antibodies in hard-to-treat brain metastases from different primary tumors that had metastasized to the brain.     

TGN1412 Induces Lymphopenia and Human Cytokine Release in a Humanized Mouse Model

Therapeutic monoclonal antibodies (mAbs) such as the superagonistic, CD28-specific antibody TGN1412, or OKT3, an anti-CD3 mAb, can cause severe adverse events including cytokine release syndrome. A predictive model for mAb-mediated adverse effects, for which no previous knowledge on severe adverse events to be expected or on molecular mechanisms underlying is prerequisite, is not available yet. We used a humanized mouse model of human peripheral blood mononuclear cell-reconstituted NOD-RAG1^-/-Aβ^-/-HLADQ^{(tg+ or tg-)}IL-2Rγc^-/- mice to evaluate its predictive value for preclinical testing of mAbs. 2–6 hours after TGN1412 treatment, mice showed a loss of human CD45⁺ cells from the peripheral blood and loss of only human T cells after OKT3 injection, reminiscent of effects observed in mAb-treated humans. Moreover, upon OKT3 injection we detected selective CD3 downmodulation on T cells, a typical effect of OKT3. Importantly, we detected release of human cytokines in humanized mice upon both OKT3 and TGN1412 application. Finally, humanized mice showed severe signs of illness, a rapid drop of body temperature, and succumbed to antibody application 2–6 hours after administration. Hence, the humanized mouse model used here reproduces several effects and adverse events induced in humans upon application of the therapeutic mAbs OKT3 and TGN1412.     

Engineering Fully Human Monoclonal Antibodies from Murine Variable Regions

Fully human monoclonal antibodies (mAbs) derived from transgenic mice or human antibody libraries are the current state of the art for reducing the immunogenicity risk of antibody drugs. Here, we describe a novel method for generating fully human mAbs from nonhuman variable regions using information from the human germline repertoire. Central to our strategy is the rational engineering of residues within and proximal to CDRs and the V_H/V_L interface by iteratively exploring substitutions to the closest human germline sequences using semi-automated computational methods. Starting from the parent murine variable regions of three currently marketed mAbs targeting CD25, vascular endothelial growth factor, and tumor necrosis factor alpha, we have generated fully human antibodies with 59, 46, and 45 substitutions, respectively, compared to the parent murine sequences. A large number of these substitutions were in the CDRs, which are typically avoided in humanization methods. Antigen affinities of the fully human variants were comparable to the chimeric mAbs in each case. Furthermore, in vitro functional characterization indicated that all retain potency of the chimeric mAbs and have comparable activity to their respective marketed drugs daclizumab, bevacizumab, and infliximab. Based on local and global sequence identity, the sequences of our engineered mAbs are indistinguishable from those of fully human mAbs isolated from transgenic mice or human antibody libraries. This work establishes a simple rational engineering methodology for generating fully human antibody therapeutics from murine mAbs produced from standard hybridoma technology.     

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.     

Expression of humanized anti-Her2/neu single-chain IgG1-like antibody in mammary glands of transgenic mice

A system for production of single-chain antibody in mammary glands of mice was developed on the basis of a hybrid gene constructed from the coding sequence of anti-Her2/neu single-chain antibody inserted into the first exon of the sheep beta-lactoglobulin gene. Lines of transgenic mice were obtained that expressed humanized single-chain anti-Her2/neu IgG1-like antibody in their milk. These antibodies interact with Her2/neu antigen with high affinity (K_d = 0.4 nM). The expression level of the transgene depended on its integration site in the genome but not on the copy number. The transgene had no toxic effect on the mice and was stably inherited, at least for two generations. The results reveal new opportunities of producing single-chain antibodies in the milk of animals.     

重组抗体药物研究进展及应用

重组抗体药物的发展经历了鼠源单克隆抗体（McAb）、人 鼠嵌合抗体、人源化抗体和全人抗体等阶段,目前初步应用于抗肿瘤、抗自身免疫病、抗感染等领域。保持和提高抗体的亲和力、降低抗体的免疫原性是抗体药物基因工程改造的两大原则。在嵌合抗体成功的基础上,通过CDR移植、表面修饰、抗体库以及转基因鼠技术,逐步提高人源化程度至100%。然而,实验室水平的研究结果与实际应用仍然存在一定差距。就重组抗体药物的基本概况、现存的问题与可能的解决办法以及在肿瘤、病毒性疾病和阿尔茨海默病治疗上的应用情况等进行了综述。     

A humanized neutralizing antibody against MERS-CoV targeting the receptor-binding domain of the spike protein

The newly-emerging Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe and fatal acute respiratory disease in humans. Despite global efforts, the potential for an associated pandemic in the future cannot be excluded. The development of effective counter-measures is urgent. MERS-CoV-specific anti-viral drugs or vaccines are not yet available. Using the spike receptor-binding domain of MERS-CoV (MERS-RBD) to immunize mice, we identified two neutralizing monoclonal antibodies (mAbs) 4C2 and 2E6. Both mAbs potently bind to MERS-RBD and block virus entry in vitro with high efficacy. We further investigated their mechanisms of neutralization by crystallizing the complex between the Fab fragments and the RBD, and solved the structure of the 4C2 Fab/MERS-RBD complex. The structure showed that 4C2 recognizes an epitope that partially overlaps the receptor-binding footprint in MERS-RBD, thereby interfering with the virus/receptor interactions by both steric hindrance and interface-residue competition. 2E6 also blocks receptor binding, and competes with 4C2 for binding to MERS-RBD. Based on the structure, we further humanized 4C2 by preserving only the paratope residues and substituting the remaining amino acids with the counterparts from human immunoglobulins. The humanized 4C2 (4C2h) antibody sustained similar neutralizing activity and biochemical characteristics to the parental mouse antibody. Finally, we showed that 4C2h can significantly abate the virus titers in lungs of Ad5-hCD26-transduced mice infected with MERS-CoV, therefore representing a promising agent for prophylaxis and therapy in clinical settings.     

Human Anti-CCR4 Minibody Gene Transfer for the Treatment of Cutaneous T-Cell Lymphoma

Background

Although several therapeutic options have become available for patients with Cutaneous T-cell Lymphoma (CTCL), no therapy has been curative. Recent studies have demonstrated that CTCL cells overexpress the CC chemokine receptor 4 (CCR4).

Methodology/Principal Findings

In this study, a xenograft model of CTCL was established and a recombinant adeno-associated viral serotype 8 (AAV8) vector expressing a humanized single-chain variable fragment (scFv)-Fc fusion (scFvFc or “minibody”) of anti-CCR4 monoclonal antibody (mAb) h1567 was evaluated for curative treatment. Human CCR4⁺ tumor-bearing mice treated once with intravenous infusion of AAV8 virions encoding the h1567 (AAV8-h1567) minibody showed anti-tumor activity in vivo and increased survival. The AAV8-h1567 minibody notably increased the number of tumor-infiltrating Ly-6G⁺ FcγRIIIa(CD16A)⁺ murine neutrophils in the tumor xenografts over that of AAV8-control minibody treated mice. Furthermore, in CCR4⁺ tumor-bearing mice co-treated with AAV8-h1567 minibody and infused with human peripheral blood mononuclear cells (PBMCs), marked tumor infiltration of human CD16A⁺ CD56⁺ NK cells was observed. The h1567 minibody also induced in vitro ADCC activity through both mouse neutrophils and human NK cells.

Conclusions/Significance

Overall, our data demonstrate that the in vivo anti-tumor activity of h1567 minibody is mediated, at least in part, through CD16A⁺ immune effector cell ADCC mechanisms. These data further demonstrate the utility of the AAV-minibody gene transfer system in the rapid evaluation of candidate anti-tumor mAbs and the potency of h1567 as a potential novel therapy for CTCL.     

Adapting antibodies for clinical use.

Techniques for antibody engineering are now overcoming the problems that have prevented monoclonal antibodies being used routinely in clinical practice. With chemical and genetic manipulation antibodies can be linked to bacterial toxins, enzymes, radionuclides, or cytotoxic drugs, allowing targeting of treatment. Antigen binding sites from antibodies raised in mice can be jointed with human IgG to reduce immunogenicity. In vitro gene amplification and genetic engineering of bacteriophage have produced large antibody gene libraries and facilitated large scale production of human monoclonal antibodies with high specificity. The trickle of monoclonal antibodies into clinical practice may soon become a flood.     

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