Monoclonal antibodies - A repertoire of therapeutics.

Humanized antibodies (Abs) are effective drugs against a variety of diseases such as cancer, autoimmune diseases, transplant rejection and others. The most powerful technology to develop humanized Abs is the use of mice that produce humanized Abs. By modifying the genetic background of F004 mice a new mouse substrain was developed for optimized "one step" generation of chimeric humanized monoclonal Abs. The new mice (F004-Jen) demonstrated improved fertility still expressing the human locus at the same level as the parental F004 mouse. The value of these mice for the generation of chimeric Abs was exemplified for a panel of chimeric Abs against the human neural cell adhesion molecule (NCAM): The fully chimeric human IgG1/kappa Ab Ch.MK1 bound to NCAM expressing cells with a K(D)=4.3-8.7 x 10(-8) M and was functionally active as demonstrated by depleting NCAM expressing cells. We also demonstrated that chimeric IgG1/kappa Abs can be induced by hybridoma class switching of IgM producing hybridoma cells, providing an alternative way to chimeric Abs. The present data highlight F004-Jen mice as an efficient tool for "one step" generation of chimeric Abs.

Assessment of the neutralizing activity of human monoclonal antibodies against HCV and also study their safety in experimental small animals (Swiss mice). Assessment of neutralizing activity of human monoclonal antibodies against HCV envelope regions (E1, E2) by two methods: by HCV cc infectious system 1) and by using positive HCV positive serum as source of HCV particles genotype 4a (neutralizing assay 2). Dot ELISA was used to study the activity of the generated antibodies. Safety and toxicity of the generated human antibodies were tested by assessing the changes in the biochemistry of liver function and kidney function tests, Complete blood counts (CBC) and studying the pathological changes with different concentrations of purified human antibodies were carried out.. Human Abs # 5 & 11 showed neutralizing activity by (neutralizing assay 2) but were not neutralizing by HCV cc assay. Human Abs # 12 & 15 showed neutralizing activity by the two methods i.e our generated human antibodies Abs# 5 &11 & 12 & 15 were neutralizing for HCV genotype 4a and Abs # 12 & 15 were neutralizing for HCV genotypes 4a and 2a. Liver and kidney functions and CBC results indicated that doses of 10 μg, 100 μg were safe. The histopathological results indicated that the dose of 10 μg of purified human monoclonal antibodies per mouse body weight was safe. The generated human monoclonal antibodies can be used to develop potent immunotherapy agents that can be administrated for the post-transplantation patients to prevent the recurrence of HCV infection. Also, the monoclonal antibodies can be used to develop a candidate vaccine against HCV.

Several human monoclonal Abs for treating Influenza have been evaluated in clinical trials with limited success despite demonstrating superiority in preclinical animal models including mice. To conduct efficacy studies in mice, human monoclonal Abs are genetically engineered to contain mouse heavy chain constant domain to facilitate the engagement of Fc-receptors on mouse immune effector cells. Although studies have consistently reported discrepancies in Ab effectiveness following genetic engineering, the structural and mechanistic basis for these inconsistencies remain uncharacterized. Here, we use homology modeling to predict variable region (VR) analogous monoclonal Abs possessing human IgG1, mouse IgG1, and mouse IgG2a heavy chain constant domains. We then examine predicted 3D structures for variations in the spatial location and orientation of corresponding paratope amino acid residues. By structurally aligning crystal structures of Fabs in complex with hemagglutinin (HA), we show that corresponding paratope amino acid residues for VR-analogous human IgG1, mouse IgG1, and mouse IgG2a monoclonal Abs interact differentially with HA suggesting that their epitopes might not be identical. To demonstrate that variations in the paratope 3D fine architecture have implications for Ab specificity and effectiveness, we genetically engineered VR-analogous human IgG1, human IgG4, mouse IgG1, and mouse IgG2a monoclonal Abs and explored their specificity and effectiveness in protecting MDCK cells from infection by pandemic H1N1 and H3N2 Influenza viruses. We found that VR-analogous monoclonal Abs placed on mouse heavy chain constant domains were more efficacious at protecting MDCK cells from Influenza virus infection relative to those on human heavy chain constant domains. Interestingly, mouse but not human heavy chain constant domains increased target breadth in some monoclonal Abs. These data suggest that heavy chain constant domain sequences play a role in shaping Ab repertoires that go beyond class or sub-class differences in immune effector recruitment. This represents a facet of Ab biology that can potentially be exploited to improve the scope and utilization of current therapeutic or prophylactic candidates for influenza.

Antigen-specific antibodies (Abs) to the 19-kDa carboxy-terminal region of Plasmodium falciparum merozoite surface protein 1 (MSP1(19)) play an important role in protective immunity to malaria. Mouse monoclonal Abs (MAbs) 12.10 and 12.8 recognizing MSP1(19) can inhibit red cell invasion by interfering with MSP1 processing on the merozoite surface. We show here that this ability is dependent on the intact Ab since Fab and F(ab')(2) fragments derived from MAb 12.10, although capable of binding MSP1 with high affinity and competing with the intact antibody for binding to MSP1, were unable to inhibit erythrocyte invasion or MSP1 processing. The DNA sequences of the variable (V) regions of both MAbs 12.8 and 12.10 were obtained, and partial amino acid sequences of the same regions were confirmed by mass spectrometry. Human chimeric Abs constructed by using these sequences, which combine the original mouse V regions with human gamma1 and gamma3 constant regions, retain the ability to bind to both parasites and recombinant MSP1(19), and both chimeric human immunoglobulin G1s (IgG1s) were at least as good at inhibiting erythrocyte invasion as the parental murine MAbs 12.8 and 12.10. Furthermore, the human chimeric Abs of the IgG1 class (but not the corresponding human IgG3), induced significant NADPH-mediated oxidative bursts and degranulation from human neutrophils. These chimeric human Abs will enable investigators to examine the role of human Fcgamma receptors in immunity to malaria using a transgenic parasite and mouse model and may prove useful in humans for neutralizing parasites as an adjunct to antimalarial drug therapy.

The mouse anti-human CD3 mAb OKT3 is a potent immunosuppressive agent used for the treatment of acute transplant rejection. OKT3 therapy is associated with acute toxicity resulting from in vivo T cell activation and systemic cytokine release, and a human anti-mouse Ab response. T cell activation is thought to be triggered by CD3 cross-linking mediated by the Abs bridging T cells and Fc receptor-bearing cells. Recent studies in a mouse model indicate that anti-mouse CD3 Abs with low affinity for Fc receptors can achieve immunosuppression without T cell activation, toxicity, or an anti-Ab response. To obtain an analogous Ab to improve the current anti-human CD3 therapy, a humanized Ab with low affinity for Fc receptors is needed. In this study, we introduced mutations into the upper CH2 region of IgG2 and expressed the altered Fc as chimeric OKT3 Abs. Compared with chimeric OKT3 IgG1, IgG2, IgG3, and IgG4, the IgG2 mutants were less mitogenic to T cells, and they did not induce the release of TNF-alpha, IFN-gamma, or IL-2. In parallel, we observed no functional interaction of the IgG2 mutant Abs with K562 cells, which express the IgG2-binding Fc receptor on their surface. Despite no measurable T cell activation, the mutant Abs could still modulate the CD3 complex. When coupled to a humanized anti-CD3, the IgG2 variant may provide a drug with less acute toxicity and immunogenicity, but may still retain potent immunosuppressive properties.

BackgroundMany cancers acquire mechanisms to evade immunosurveillance by activating immune checkpoint pathways, which suppress the antitumor immune responses. Monoclonal antibodies (ab’s) targeting immune checkpoints, such as CTLA-4 and PD-1, have shown excellent results in several cancers and are currently being investigated in clinical trials for various malignancies. The clinically tested a-CTLA-4 (Ipilimumab) and a-PD-1 (Nivolumab and Pembrolizumab) ab’s are fully human or humanized ab’s, respectively. However, most studies conducted in mice utilize a xenogeneic a-PD-1 ab originating from rat, IgG2a RMP1-14 clone. This has been proposed to cause adverse effects in the commonly used 4T1 mammary carcinoma model of triple negative breast cancer (TNBC). Repeated administration of xenogeneic a-PD-1 ab’s in this model results in fatal hypersensitivity reactions in tumor bearing mice, and unlike human TNBC, the 4T1 cell line is generally poorly responsive to immune checkpoint inhibitors. Recently, a semi-syngeneic recombinant a-PD-1 ab has been developed by transferring the variable regions of RMP1-14 onto a murine IgG1e3 constant region.Materials and MethodsTesting xenogeneic and semi-syngeneic a-PD-1 ab with and without a-CTLA-4 ab in BALB/c mice carrying 4T1 luciferase positive tumors.ResultsIn this study, we compared a semi-syngeneic recombinant a-PD-1 ab to the original xenogeneic RMP1-14 clone for treatment of luciferase positive 4T1 carcinomas. Surprisingly, the semi-syngeneic a-PD-1 ab was not able to circumvent the fatal hypersensitivity reactions. Still, the combination therapy of a-CTLA-4 and the semi-syngeneic a-PD-1 ab significantly reduced tumor volume in 4T1-luciferase tumor bearing mice compared to isotype control-treated mice already from day 16 post tumor inoculation (day 8 post treatment-initiation). In contrast, xenogeneic a-PD-1/a-CTLA-4 treated mice did not show significant difference from the control group until 24 days post tumor inoculation and never to the same degree. Furthermore, analysis of the T cell responses towards the murine tumor-associated antigen AH-1, revealed that treatment with syngeneic a-PD-1/a-CTLA-4 ab gave a significantly stronger CD8+ T cell response over both control mice and mice treated with xenogeneic a-PD-1/a-CTLA-4 ab.ConclusionsThese studies indicate that the semi-syngeneic a-PD-1 IgG1e3 ab might be a more efficient and translatable a-PD-1 ab for preclinical in vivo studies, which is important for the future investigation of immune checkpoint inhibitor therapy.Disclosure InformationI. Skandorff Pedersen: A. Employment (full or part-time); Significant; InProTher Aps. K. Orfin: A. Employment (full or part-time); Significant; InProTher Aps. K.N. Nielsen: A. Employment (full or part-time); Significant; InProTher Aps. P.J. Holst: A. Employment (full or part-time); Significant; InProTher Aps. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; InProTher Aps.

Inhibition of T cell activation with a humanized anti-β1 integrin chain mAb

Background and aims Anti-idiotypic antibodies (Abs) represent an alternative vaccination approach in human therapy. This approach is based on the idiotype (Id) network theory postulated by Jerne describing the Ab (Ab1) – anti-idiotypic Ab (Ab2) – anti-anti-idiotypic Ab (Ab3) cascade stimulation. Specific anti-Id Abs serve as an “internal image” of the target antigen and can be used to induce Abs able to bind to the cognate antigen [1]. The anti-Id Ab Ab2/ 3H6 [2], developed at our Institute was generated in mouse and is directed against the human monoclonal Ab (mAb) 2F5, which broadly and potently neutralizes primary HIV-1 isolates [3]. Ab2/3H6 which has been characterized previously [4,5] is able to mimic the antigen recognition site of 2F5 and therefore it is suggested as a putative candidate for an HIV-1 vaccine. We investigated the potential of Ab2/3H6 by immunization of Fab fragments and fusion proteins with interleukin 15 (IL15) and tetanus toxin (TT) tags as immune modulators. After three prime/boost administrations rabbit sera were purified and analyzed for 2F5-like specific Abs. Further, the 2F5-like Abs from the sera were enriched by affinity purification and characterized for their binding affinity to 2F5. In an additional approach we applied different humanization methods to reduce the immunogenicity of the originally mouse derived Ab2/3H6. The mouse variable regions of Ab2/3H6 were subjected to three different humanization methods, namely resurfacing, CDR-grafting and superhumanization. Four differently humanized Ab2/3H6 variants were characterized for their binding affinity to 2F5 in comparison to the original Ab2/3H6. Results To evaluate the humoral immune response of Ab2/3H6 we designed Ab2/3H6 fusion proteins with IL15 and TT. Recombinant CHO cell lines were established and after protein purification New Zealand white rabbits were immunized with the Ab2/3H6 variants. Ten days after the final boost sera were collected and analyzed for total rabbit IgG levels. After proteinA affinity purification of the sera the isolated rabbit IgGs were tested for Ab2/3H6 and recombinant gp140 (UG37) specificity (Figure 1A). Further an affinity enrichment step using a UG37/ELDKWA column was performed and the obtained Ab3 fraction was tested on UG37 (Figure 1B) and additionally on the original 2F5 epitope ELDKWA (Figure 1C). Finally the Ab3 fraction was tested for binding affinity to the UG37 in a bio-layer interferometry assay which showed that the Ab3 fraction has a 6.6 fold reduced affinity towards UG37 compared to the mAb 2F5 (Table 1). For the humanization approach three different methods were chosen. The “resurfaced” variant (RS3H6) was developed by a computer model and surface exposed amino acids in the murine framework (FR) were substituted by residues usually found at equivalent positions in human Abs. The “superhumanized” form (SH3H6) was designed by structural homologies between the murine Ab2/3H6 CDRs and human germline CDRs. The most homologous human germline Ab was then used as acceptor FR. For the “CDR-grafted” variant two versions were expressed. An “aggressive” graft (GA3H6) harbouring less backmutations, making the grafted Ab more human-like and a “conservative” graft (GC3H6) with more backmutations. The different “reshaped” variable regions of Ab2/3H6 were expressed in CHO-cells as IgG1 molecules. The obtained “humanized” Ab variants were further characterized by competition ELISA (Figure 1D). * Correspondence: renate.kunert@boku.ac.at Department of Biotechnology, Institute for Applied Microbiology, BOKU – University of Natural Resources and Life Sciences, A-1190 Vienna, Austria Kunert and Mader BMC Proceedings 2011, 5(Suppl 8):P64 http://www.biomedcentral.com/1753-6561/5/S8/P64

Constant (C)-region switching of heavy (H) and/or light (L) chains in antibodies (Abs) can affect their affinity and specificity, as demonstrated using mouse, human, and chimeric mouse-human (MH) Abs. However, the consequences of C-region switching between evolutionarily distinct mammalian and avian Abs remain unknown. To explore C-region switching in mouse-chicken (MC) Abs, we investigated antigen-binding parameters and thermal stability of chimeric MC-6C407 and MC-3D8 IgY Abs compared with parental mouse IgGs and chimeric MH Abs (MH-6C407 IgG and MH-3D8 IgG) bearing identical corresponding variable (V) regions. The two MC-IgYs exhibited differences in antigen-binding parameters and thermal stability from their parental mouse Abs. However, changes were similar to or less than those between chimeric MH Abs and their parental mouse Abs. The results demonstrate that mammalian and avian Abs share compatible V-C region interfaces, which may be conducive for the design and utilization of mammalian-avian chimeric Abs.

The anti-idiotypic antibody (Ab) Ab2/3H6 (Kunert et al. 2002) is directed against the human monoclonal Ab (mAb) 2F5, which broadly and potently neutralizes primary HIV-1 isolates (Wolbank et al. 2003). Ab2/3H6 is able to mimic the antigen recognition site of the mAb2F5 making it an attractive candidate antigen for HIV-1 vaccine purposes. In this study the mouse variable regions of Ab2/3H6 were subjected to various humanizing approaches using three different methods. For the CDR-grafted variants, an “aggressive” graft harboring less backmutations and a “conservative” graft with more backmutations were designed. In the Superhumanization approach we grafted the murine CDRs to a human framework (FR) which was most related concerning the canonical structure class. The Resurfacing method substitutes mouse amino acids (aa) that are surface exposed in the murine FR by residues usually found in equivalent positions in human Abs. The different Ab2/3H6 variants were characterized by competition experiments with mAb 2F5. The resurfaced and the “conservative” grafted variants showed similar binding properties to mAb 2F5 when compared to the murine Ab2/3H6 version, while the “aggressive” grafted Ab showed less affinity and the superhumanized type lost the ability to bind to mAb 2F5.

High-Throughput Screening of a Large Combinatorial Library of Small Proteins Against Human Spleen-Derived Monoclonal Anti-ADAMTS13 Antibodies Yields Specific Anti-Idiotypic Molecules – Potential Tools for New Treatment Strategy in Acquired TTP

Both polyclonal and monoclonal human antibodies (Abs) to the V3 domain of HIV-1 gp120 display cross-clade neutralizing activity against primary isolates and T cell-adapted virus strains. The most broadly neutralizing of the human anti-V3 monoclonal Abs (mAbs), 447-52D, recognizes 14 amino acids, including the GPxR core epitope at the tip of the V3 loop. Monoclonal Ab 447-52D neutralized 92% of 38 primary isolates carrying the GPGR V3 motif regardless of whether the viruses belonged to clades A, B, F, or H; in contrast, none of 19 viruses with the GPGQ and other non-GPGR/Q sequences at the tip of the V3 loop was sensitive to mAb 447-52D. These data are consistent with the crystallographic resolution of a complex of the Fab fragment of mAb 447-52D with a V3 peptide that shows that the binding specificity of the mAb is due to recognition of the GPGR motif at the tip of the loop. The critical role of the Arg residue in this motif was determined using viruses pseudotyped with the envelope of primary isolate CA1 containing the GPGR motif or with a mutated envelope with a Gln (Q) replacing the Arg (R) at the tip of the loop. While the wild-type pseudovirus was neutralized by mAb 447-52D, the pseudovirus carrying the point mutation was resistant to neutralization. These data illuminate the structural basis for both the breadth and specificity of a broadly neutralizing human mAb and contribute to our understanding of the epitopes recognized by Abs that protect against infection with HIV-1.

Both polyclonal and monoclonal human antibodies (Abs) to the V3 domain of HIV-1 gp120 display cross-clade neutralizing activity against primary isolates and T cell-adapted virus strains. The most broadly neutralizing of the human anti-V3 monoclonal Abs (mAbs), 447-52D, recognizes 14 amino acids, including the GPxR core epitope at the tip of the V3 loop. Monoclonal Ab 447-52D neutralized 92% of 38 primary isolates carrying the GPGR V3 motif regardless of whether the viruses belonged to clades A, B, F, or H; in contrast, none of 19 viruses with the GPGQ and other non-GPGR/Q sequences at the tip of the V3 loop was sensitive to mAb 447-52D. These data are consistent with the crystallographic resolution of a complex of the Fab fragment of mAb 447-52D with a V3 peptide that shows that the binding specificity of the mAb is due to recognition of the GPGR motif at the tip of the loop. The critical role of the Arg residue in this motif was determined using viruses pseudotyped with the envelope of primary isolate CA1 containing the GPGR motif or with a mutated envelope with a Gln (Q) replacing the Arg (R) at the tip of the loop. While the wild-type pseudovirus was neutralized by mAb 447-52D, the pseudovirus carrying the point mutation was resistant to neutralization. These data illuminate the structural basis for both the breadth and specificity of a broadly neutralizing human mAb and contribute to our understanding of the epitopes recognized by Abs that protect against infection with HIV-1.

Introduction:Monoclonal antibody is an antibody that is specific for one antigen and is produced by a B-cell hybridoma. Hybridoma technology has been replaced by recombinant DNA technology and transgenic method (mice, plant and chickens). Once monoclonal antibodies for a given substance have been produced, they can be used to detect the presence and quantity of this substance and so monoclonal antibody has many diagnostic applications. Aim of the work:To clarify the new methods for production of monoclonal antibodies and application of monoclonal antibodies in diagnostic and therapeutic approaches. Conclusion:Hybridoma technology has been replaced by recombinant DNA technology and transgenic method (mice, plant and chickens). Once monoclonal antibodies for a given substance have been produced, they can be used to detect the presence and quantity of this substance and so monoclonal antibody has many diagnostic applications as in the analysis of cell surface and secreted molecules like CD molecules identification and identification of transcription factor FOXP3. Also, they have a role in the molecular genomics to identify the susceptible genes that predispose to development of autoimmune thyroid diseases.

Specific detection of antibodies in cancer patients following immunotherapy with anti-idiotype