Anti-CD30 (Ber-H2) epitope requires structural elements as shown by mass spectroscopy and dual-site associated kinetics.

The Ber-H2 mouse monoclonal antibody has been in use for 35 years for detecting the CD-30 biomarker in a variety of lymphomas. Despite the wide use of this clone, we have not been successful in applying synthetic peptides derived from the published epitope sequence and affinity data toward the development of a new Ber-H2-based in vitro diagnostic reagent assay. We found that synthetic peptides based on the published epitope sequence do not function to inhibit antibody-binding activity, thus indicating that the sequence is not the full epitope recognized by Ber-H2. In this report, we used mass spectroscopic analysis of proteolyzed CD30 fragments capable of binding Ber-H2 to identify additional regions within the epitope that participate in binding. Using surface plasmon resonance binding kinetic analyses and immuno-histochemical peptide-inhibition assays, we also demonstrate that the epitope sequence as originally reported is missing two key elements necessary for binding the Ber-H2 antibody.

Increased HIV-1 transcriptional activity and infectious burden in peripheral blood and gut-associated CD4+ T cells expressing CD30.

HIV-1-infected cells persist indefinitely despite the use of combination antiretroviral therapy (ART), and novel therapeutic strategies to target and purge residual infected cells in individuals on ART are urgently needed. Here, we demonstrate that CD4+ T cell-associated HIV-1 RNA is often highly enriched in cells expressing CD30, and that cells expressing this marker considerably contribute to the total pool of transcriptionally active CD4+ lymphocytes in individuals on suppressive ART. Using in situ RNA hybridization studies, we show co-localization of CD30 with HIV-1 transcriptional activity in gut-associated lymphoid tissues. We also demonstrate that ex vivo treatment with brentuximab vedotin, an antibody-drug conjugate (ADC) that targets CD30, significantly reduces the total amount of HIV-1 DNA in peripheral blood mononuclear cells obtained from infected, ART-suppressed individuals. Finally, we observed that an HIV-1-infected individual, who received repeated brentuximab vedotin infusions for lymphoma, had no detectable virus in peripheral blood mononuclear cells. Overall, CD30 may be a marker of residual, transcriptionally active HIV-1 infected cells in the setting of suppressive ART. Given that CD30 is only expressed on a small number of total mononuclear cells, it is a potential therapeutic target of persistent HIV-1 infection.

Anaplastic large cell lymphoma and lepromatous leprosy: a rare coexistence.

Lepromatous leprosy (LL) has been reported in the literature with Non Hodgkin Lymphoma and rarely with Hodgkin Lymphoma. However, an extensive search of the literature shows no case report describing anaplastic large cell lymphoma (ALCL) in association with LL. We report a case of a young male with LL who was found to have ALCL. This is an interesting case of coexistence of an endemic infectious disease and a rare lymphoma involving the same lymph node, with a brief review of the literature.

Antibody fusion proteins with human ribonucleases 1 to 8.

ImmunoRNases combine tumor targeting by antibodies with the cytotoxic action of ribonucleases from the RNase A superfamily. This study investigated for the first time all catalytic active human RNase A family members (1 to 8) as effector components of antibody fusion proteins. ImmunoRNase fusion proteins were constructed using the CD30-specific bivalent recombinant scFv-Fc antibody SH313-B5. Production of the resulting entirely human immunoRNases 1 to 8 was done in mammalian cells by secretion of active forms. The immunoRNases mediated CD30-specific cell binding and showed ribonucleolytic activity. Interestingly, immunoRNases 1 and 2 were active in the presence of up to 5-/20-fold molar excess of the pancreatic RNase inhibitor (RI), which is supposed to efficiently inhibit all human RNase A activity. ImmunoRNases 3, 4, 6 and 7 were only inhibited by several fold molar excess of RI, whereas immunoRNases 5 and 8 were already completely inactive at equimolar RI concentrations. Compared to free RNases, activity and RI sensitivity were not significantly changed by antibody fusion or dimerisation. ImmunoRNase3 and 5 mediated tumor growth inhibition at low nanomolar concentrations. Anti-tumor activity was antigen-specific and did not show any correlation with ribonucleolytic activity or RI sensitivity.

A novel enediyne-integrated antibody-drug conjugate shows promising antitumor efficacy against CD30+ lymphomas.

CD30 is a 120-kDa type I transmembrane glycoprotein belonging to the tumor necrosis factor receptor superfamily. Overexpression of CD30 has been reported in Hodgkin's lymphoma (HL) and anaplastic large-cell lymphoma (ALCL). CD30-targeted treatment with antibody-drug conjugates (ADCs) can lead to promising clinical benefit. Lidamycin (LDM), consisting of an apoprotein LDP and an active enediyne chromophore AE, is a member of the enediyne antibiotic family and one of the most potent antitumor agents. AE and LDP can be dissociated and reconstituted under certain conditions in vitro. LDM is an ideal payload for the preparation of ADCs. In this study, we show the generation, production, and antitumor activity of anti-CD30-LDM, a novel ADC which consists of the intact anti-CD30 antibody and LDM. First, the anti-CD30-LDP fusion protein was constructed and expressed in CHO/dhFr- cells. Anti-CD30-LDP showed specific and high-affinity binding to CD30 and could be internalized into target cells. It also exhibited excellent tumor-targeting capability in vivo. Next, anti-CD30-LDM was prepared by assembling the enediyne molecule AE to the fusion protein anti-CD30-LDP. Anti-CD30-LDM was highly cytotoxic to HL and ALCL cell lines, with IC50 values of 5-50 pm. It can also induce cell apoptosis and G2/M cell cycle arrest. In the Karpas299 xenograft model, the tumor growth was inhibited by 87.76% in mice treated with anti-CD30-LDM and with no discernible adverse effects. Taken together, anti-CD30-LDM shows attractive tumor-targeting capability and antitumor efficacy both in vitro and in vivo and could be a promising candidate for the treatment of CD30+ lymphomas.

New anti-CD30 human pancreatic ribonuclease-based immunotoxin reveals strong and specific cytotoxicity in vivo.

Although the therapy of Hodgkin lymphoma and anaplastic large cell lymphoma has been considerably improved during the last decades, high therapeutic toxicity, relapses, secondary tumors, and primary treatment failure(s) occur. Both malignancies are well suited for CD30-targeted immunotherapy because of their strong CD30 expression. We constructed an immunotoxin composed of a single chain variable fragment of a CD30 antibody fused to the human pancreatic ribonuclease, showing CD30-specific binding and ribonucleolytic activity resistant to the inhibitor RNasin. This immunotoxin revealed CD30-specific anti-tumor activity in BALB/c mice that were challenged with CD30-positive or CD30-negative syngeneic tumor cells.

Diagnostic, prognostic and therapeutic role of CD30 in lymphoma.

INTRODUCTION: CD30 is a cell surface receptor expressed in classical Hodgkin lymphoma (HL), anaplastic large cell lymphoma (ALCL), and many other lymphomas to a variable degree. It has been identified as an important therapeutic target in lymphoma. Areas covered: CD30 testing is essential in diagnosis of classical HL and ALCL, and expression can also be seen in other lymphoma subtypes. Development of Brentuximab vedotin (BV), an antibody-drug conjugate directed to CD30, has been an important advance in lymphoma treatment. It is approved in treatment of relapsed HL and ALCL, as well as post-transplant maintenance for HL, and has been shown to be effective in other CD30-expressing lymphomas. This review describes the role of CD30 and the use of CD30-targeted agents in HL, ALCL, and other lymphomas, including review of relevant trials of BV. Expert commentary: Recognition of CD30 expression in lymphoma has led to the development of important therapeutic options. Multiple trials are ongoing combining BV with other agents, such as chemotherapy or immunotherapy, to develop more effective regimens. In addition, treatments targeting CD30 in different ways are being developed, such as bispecific antibodies and chimeric antigen receptor (CAR) T-cells.

Highly Potent, Anthracycline-based Antibody-Drug Conjugates Generated by Enzymatic, Site-specific Conjugation.

Antibody-drug conjugates (ADC) are highly potent and specific antitumor drugs, combining the specific targeting of mAbs with the potency of small-molecule toxic payloads. ADCs generated by conventional chemical conjugation yield heterogeneous mixtures with variable pharmacokinetics, stability, safety, and efficacy profiles. To address these issues, numerous site-specific conjugation technologies are currently being developed allowing the manufacturing of homogeneous ADCs with predetermined drug-to-antibody ratios. Here, we used sortase-mediated antibody conjugation (SMAC) technology to generate homogeneous ADCs based on a derivative of the highly potent anthracycline toxin PNU-159682 and a noncleavable peptide linker, using the anti-HER2 antibody trastuzumab (part of Kadcyla) and the anti-CD30 antibody cAC10 (part of Adcetris). Characterization of the resulting ADCs in vitro and in vivo showed that they were highly stable and exhibited potencies exceeding those of ADCs based on conventional tubulin-targeting payloads, such as Kadcyla and Adcetris. The data presented here suggest that such novel and highly potent ADC formats may help to increase the number of targets available to ADC approaches, by reducing the threshold levels of target expression required. Mol Cancer Ther; 16(5); 879-92. ©2017 AACR.

An anti-CD30 chimeric receptor that mediates CD3-zeta-independent T-cell activation against Hodgkin's lymphoma cells in the presence of soluble CD30.

Hodgkin's lymphoma patients fail to establish an efficient cellular response against CD30+ Hodgkin/Reed-Sternberg cells. An impaired T-cell receptor/CD3-zeta-mediated activation of T cells is thought to be involved in this situation. We here present a chimeric anti-CD30 receptor that mediates MHC and T-cell receptor/CD3-zeta-independent T-cell activation against CD30+ lymphoma cells even in the presence of soluble CD30. The receptor consists of the binding domain of the monoclonal antibody HRS3 and the signaling unit of the Fc epsilonRI-receptor gamma-chain. After expression in MD45 T cells, receptor cross-linking with immobilized anti-idiotypic monoclonal antibody and CD30+ cells, respectively, results in increased interleukin 2 secretion and specific cytolysis of CD30+ Hodgkin's lymphoma cells. Soluble CD30 in concentrations up to 6000 units/ml did not interfere with cellular activation induced by membrane-bound antigen. This demonstrates the feasibility of the chimeric anti-CD30-scFv-gamma receptor in CD30+ lymphoma cell targeting, even in the presence of as high concentrations of soluble CD30 as are found in patients during progression of the disease.

Ligand-independent signaling by overexpressed CD30 drives NF-kappaB activation in Hodgkin-Reed-Sternberg cells.

Overexpression of CD30 and constitutive NF-kappaB activation characterizes tumor cells of Hodgkin's disease (HD), Hodgkin and Reed-Sternberg (H-RS) cells. We report that in H-RS cells overexpression of CD30 leads to self-aggregation, recruitment of TRAF2 and TRAF5, and NF-kappaB activation, independent of CD30 ligand. CD30 and TRAF proteins co-localized in H-RS cell lines and in lymph nodes of HD. An adenovirus-vector carrying a decoy CD30 lacking the cytoplasmic region or a dominant negative IkappaBalpha mutant blocks NF-kappaB activation, down regulates IL-13 expression and induces apoptosis. Thus, in H-RS cells, ligand-independent activation of CD30 signaling drives NF-kappaB activation and this leads to constitutive cytokine expression, which provides a molecular basis for HD. Inhibition of NF-kappaB activation by adenovirus vector-mediated gene transfer may provide a novel strategy of cell- and target molecule-specific therapy for patients with HD.

The ectodomain shedding of CD30 is specifically regulated by peptide motifs in its cysteine-rich domains 2 and 5.

Tumor necrosis factor (TNF)-alpha converting enzyme (TACE) is responsible for the ectodomain release of various membrane proteins by proteolytic cleavage in close proximity to the cell membrane. Despite the wide spectrum of possible substrates, selective cleavage can be achieved by substrate cross-linking. To explore the underlying mechanism, we studied the TACE-mediated shedding of CD30. Whereas the constitutive release of the soluble ectodomain of CD30 (sCD30) from the lymphoma cell line Karpas 299 was enhanced by most anti-CD30 antibodies, it was inhibited by antibodies Ber-H2 and Ki-4. On the basis of the recognized epitopes, shedding seemed to depend on the availability of the cysteine-rich domains (CRD) 2 and 5 of the CD30 ectodomain. CRD2 and 5 have almost identical amino acid sequences and are localized distant from the TACE-targeted cleavage site. Soluble CD30, the product of this enzyme reaction, did not inhibit, but on the contrary, it stimulated CD30 shedding in a CRD2/5-dependent manner. This process could also be induced by CRD2/5-derived peptides but not by a CRD1-derived control peptide. This example of a product-activation was CD30 selective since other TACE substrates such as TNFR1 or TNF-alpha were not affected. These data suggest that CD30 shedding is stimulated by an elevated local availability of CRD2 or 5, possibly by forming a docking station for the releasing enzyme through substrate aggregation.

CD30 and type 2 T helper (Th2) responses.

CD30 is one of the members of the tumor necrosis factor receptor superfamily, originally described as a marker of Reed-Sternberg and Hodgkin's cells in Hodgkin's lymphoma. CD30 appears to be preferentially expressed on, and its soluble form (sCD30) released by, CD4+ and CD8+ T cell clones capable of producing T helper 2 (Th2)-type cytokines. In noneoplastic conditions, CD30+ T cells are barely detectable in vivo; however, a few allergen-specific CD4+CD30+ T cells inducible to the production of Th2-type cytokines could be sorted out from the circulation of allergic subjects after allergen exposure. Moreover, high numbers of CD30+ T cells were found in the lymph node of a patient suffering from Omenn's syndrome, a rare congenital Th2-mediated immunodeficiency disorder. More importantly, high serum levels of sCD30 were observed in some conditions in which a pathogenetic role for Th2 cells has been suggested, such as Omenn's syndrome, atopy, systemic lupus erythematosus, and after infection with measles virus or human immunodeficiency virus. Thus, detection of CD30+ T cells and/or of increased levels of sCD30 may reflect the presence of immune responses or immune alterations characterized by the prevalent activation of Th2-like cells.

Opposite effects of the CD30 ligand are not due to CD30 mutations: results from cDNA cloning and sequence comparison of the CD30 antigen from different sources.

CD30L, the ligand for the activation antigen CD30, is a member of the tumor necrosis factor family of cytokines. Binding of CD30L to CD30, which is a member of the nerve growth factor/tumor necrosis factor receptor family, induces proliferation in peripheral blood lymphocytes and Hodgkin's derived cell lines with a T-cell phenotype such as HDLM-2 and L540, while cell lines derived from anaplastic large cell lymphomas, such as Karpas 299, undergo cell death. In order to investigate whether mutations of the CD30 antigen are responsible for these opposite effects, we cloned the open reading frame of CD30 cDNAs from the cell lines L540 and Karpas 299 and from peripheral blood lymphocytes by reverse transcriptase polymerase chain reaction. Sequencing of independent plasmid clones revealed that these cells have a silent transition (A-->G) at position 771 of the open reading frame compared to the published sequence derived from the HTLV-1+ cell line HUT-102. As published data have shown that crosslinking of CD30 induces an elevation of cytosolic free calcium ([Ca2+]i) in TCR positive Jurkat cells, we have analysed the effect of crosslinking of CD30 on L540 and Karpas 299 cells. No elevations of [Ca2+]i have been observed in these cell lines after crosslinking of CD30 with HRS-4. We conclude (i) that the different functional effects of CD30 in PBL, L540 and Karpas 299 are not due to differences in the primary structure of the receptor; and (ii) that the different responses observed upon engagement with CD30L for the cell lines L540 and Karpas 299 do not correlate with differences in mobilization of [Ca2+]i after crosslinking of CD30.

Receptor-targeted lentiviral vectors are exceptionally sensitive toward the biophysical properties of the displayed single-chain Fv.

An increasing number of applications require the expression of single-chain variable fragments (scFv) fusion proteins in mammalian cells at the cell surface membrane. Here we assessed the CD30-specific scFv HRS3, which is used in immunotherapy, for its ability to retarget lentiviral vectors (LVs) to CD30 and to mediate selective gene transfer into CD30-positive cells. Fused to the C-terminus of the type-II transmembrane protein hemagglutinin (H) of measles virus and expressed in LV packaging cells, gene transfer mediated by the released LV particles was inefficient. A series of point mutations in the scFv framework regions addressing its biophysical properties, which substantially improved production and increased the melting temperature without impairing its kinetic binding behavior to CD30, also improved the performance of LV particles. Gene transfer into CD30-positive cells increased ∼100-fold due to improved transport of the H-scFv protein to the plasma membrane. Concomitantly, LV particle aggregation and syncytia formation in packaging cells were substantially reduced. The data suggest that syncytia formation can be triggered by trans-cellular dimerization of H-scFv proteins displayed on adjacent cells. Taken together, we show that the biophysical properties of the targeting ligand have a decisive role for the gene transfer efficiency of receptor-targeted LVs.

Cloning and characterization of a cDNA for rat CD30 homolog and chromosomal assignment of the genomic gene.

CD30 is a member of the tumor necrosis factor receptor superfamily, which is expressed on some activated lymphocytes, virus-infected cells and transformed lymphocytes. To facilitate our understanding of biological functions and functional domains, we isolated rat cDNA clones encoding the rat homolog of human CD30 from a cDNA library of a rat T-cell line, TARL-2. The nucleotide sequence of the cDNA showed 73% homology with that of human CD30. The deduced rat CD30 protein consisted of 493 amino acids with an M(r) of 59 160 and contained a single transmembrane domain. It lacked the second repeat of the cysteine-rich motif in the extracellular domain found in human CD30. The amino acid sequence showed 51.8 and 61.2% identity with the cysteine-rich and the cytoplasmic domains, respectively. In the cytoplasmic domain, however, the amino acid sequence was highly conserved in about 100 residues near the C-terminus showing 77.7% identity, whereas the rest of the cytoplasmic domain showed 45.2% identity. This conservation suggests the functional importance of this region. Comparison with the recently reported mouse CD30 revealed 83.7% conservation of the amino acid sequence and a common structure of the extracellular domain which lacks the second cysteine-rich motif. Northern blots revealed a 3.4-kb mRNA in the PHA-activated spleen cells and human T-cell leukemia virus type 1 (HTLV-1)-infected rat T-cell lines, whereas smaller transcripts of 2.3 kb were found in the lung. A rabbit polyclonal antibody raised against GST-fusion protein of the cytoplasmic domain detected bands with an apparent M(r) of 80 kDa and 100- 110 kDa expressed in TARL-2 and spleen cells. Transient overexpression of rat CD30 in TARL-2 cells activated HIV LTR in a NF-kappa B site-dependent manner, indicating that CD30 signals activate NF-kappa B. The chromosomal location of the gene was identified by fluorescence in situ hybridisation at 5q36.2, and appeared to correspond to human 1p36, where human CD30 has been mapped. The identification and characterization of the rat counterpart of human CD30 will facilitate studies of the biological function of this molecule.

Characterization of a new family of proteins that interact with the C-terminal region of the chromatin-remodeling factor CHD-3.

The two human proteins Ki-1/57 and CGI-55 have highly similar amino acid sequences but their functions are unknown. We analyzed them by yeast two-hybrid screens and found that they interact with the C-terminal region of the human chromatin-remodeling factor CHD-3 (chromo-helicase-DNA-binding domain protein-3). The interaction of CGI-55 and CHD-3 could be confirmed in vitro and in vivo by co-immunoprecipitations from Sf9 insect cells. Mapping showed that CGI-55 interacts with CHD-3 via two regions at its N- and C-terminals. The CGI-55 and Ki-1/57 mRNAs show highest expression in muscle, colon and kidney. A CGI55-GFP fusion protein was localized in the cytoplasm, nucleus and perinuclear regions of HeLa cells. These data suggest the possibility that CGI-55 and Ki-1/57 might be involved in nuclear functions like the remodeling of chromatin.

Functional humanization of an anti-CD30 Fab fragment for the immunotherapy of Hodgkin's lymphoma using an in vitro evolution approach.

CD30, the so-called Reed-Sternberg antigen, constitutes a promising cell-specific target for the treatment of Hodgkin's lymphoma. Starting from the previously characterized cognate HRS3 mouse monoclonal antibody, the bacterially produced functional Fab fragment was humanized by grafting the CDRs from the mouse antibody framework on to human immunoglobulin consensus sequences. This procedure led to a 10-fold decreased antigen affinity, which surprisingly was found to be mainly due to the VH domain. To improve the antigen-binding activity, an in vitro evolution strategy was employed, wherein random mutations were introduced into the humanized VH domain by means of error-prone PCR, followed by a filter sandwich Escherichia coli colony screening assay for functional Fab fragments using a recombinant extracellular domain of the CD30 antigen. After three cycles of in vitro affinity maturation, the optimized Fab fragment huHRS3-VH-EP3/1 was identified, which carried four exchanged residues within or close to the VH CDRs and had an affinity that was almost identical with that of the murine HRS3 Fab fragment. The resulting humanized Fab fragment was fully functional with respect to CD30 binding both in ELISA with the recombinant antigen and in FACS experiments with CD30-positive L540CY cells. In the light of the previously successful clinical application of an alphaCD30 x alphaCD16 bispecific mouse quadroma antibody derived from HRS3, the humanized Fab fragment comprises an important step towards the construction of a fully recombinant therapeutic agent. The combination of random mutagenesis and colony filter screening assay that was successfully applied here should be generally useful as a method for the rapid functional optimization of humanized antibody fragments.

Strong human leukocyte antigen matching effect in nonsensitized kidney recipients with high pretransplant soluble CD30.

The influence of human leukocyte antigen (HLA) matching on graft survival is greater in patients with preformed lymphocytotoxic antibodies than in nonsensitized patients. Pretransplant serum soluble CD30 (sCD30) affects graft outcome independently of presensitization status. The impact of HLA compatibility on kidney transplant survival was analyzed in 3980 nonsensitized first cadaveric kidney recipients in relation to the pretransplant serum sCD30 content. Although HLA compatibility influenced graft outcome only marginally in nonsensitized recipients with low sCD30 (at 3 years: P=0.0095; at 5 years: P=0.1033), a strong HLA matching effect was observed in nonsensitized recipients with high sCD30 (at 3 years: P<0.0001; at 5 years: P=0.0001). Nonsensitized patients with high pretransplant sCD30 benefit from an HLA well-matched kidney. Patients should be tested for sCD30 while on the waiting list for a kidney transplant, and HLA well-matched kidneys should be allocated to patients with high sCD30.

Different sensitivity of CD30+ cell lines to Ber-H2/saporin-S6 immunotoxin.

The in vitro sensitivity of cells to a Ber-H2(anti-CD30)/saporin-S6 immunotoxin has been investigated. The CD30+ cell lines, K562, L428 and L540, were used to study cell binding, uptake and degradation of the immunotoxin. K562 cells were less sensitive than L428 and L540 cells to the immunotoxin by approximately one order of magnitude. The difference in cytotoxicity correlated with the intracellular accumulation and with the ratio of degraded over total internalized Ber-H2/saporin-S6, regardless of the immunotoxin binding to the cells. After 6 h incubation, the less sensitive K562 cells (i) accumulated only one third and one tenth of the immunotoxin accumulated by the more sensitive L428 and L540 cells, respectively, and (ii) degraded two thirds of the internalized protein versus one third degraded by either L428 or L540 cells. Ammonium chloride and chloroquine reduced the cytotoxicity of the immunotoxin towards K562 but not to L540 cells. This effect correlated with the increment of immunotoxin catabolism by K562 cells in the presence of chloroquine. In conclusion, uptake alone of an immunotoxin by target cells is not sufficient to assure its efficacy which might also depend on intracellular routing. Only a cytotoxicity test may be really predictive.

The influence of antibody fragment format on phage display based affinity maturation of IgG.

Today, most approved therapeutic antibodies are provided as immunoglobulin G (IgG), whereas small recombinant antibody formats are required for in vitro antibody generation and engineering during drug development. Particularly,single chain (sc) antibody fragments like scFv or scFab are well suited for phage display and bacterial expression, but some have been found to lose affinity during conversion into IgG.In this study, we compared the influence of the antibody format on affinity maturation of the CD30-specific scFv antibody fragment SH313-F9, with the overall objective being improvement of the IgG. The variable genes of SH313-F9 were randomly mutated and then cloned into libraries encoding different recombinant antibody formats, including scFv, Fab, scFabΔC, and FabΔC. All tested antibody formats except Fab allowed functional phage display of the parental antibody SH313-F9, and the corresponding mutated antibody gene libraries allowed isolation of candidates with enhanced CD30 binding. Moreover, scFv and scFabΔC antibody variants retained improved antigen binding after subcloning into the single gene encoded IgG-like formats scFv-Fc or scIgG, but lost affinity after conversion into IgGs.Only affinity maturation using the Fab-like FabΔC format, which does not contain the carboxy terminal cysteines, allowed successful selection of molecules with improved binding that was retained after conversion to IgG. Thus, affinity maturation of IgGs is dependent on the antibody format employed for selection and screening. In this study, only FabΔC resulted in the efficient selection of IgG candidates with higher affinity by combination of Fab-like conformation and improved phage display compared with Fab.