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4–1BB (CD137) is an activation-induced costimulatory receptor that regulates immune responses of activated CD8 T and natural killer cells, by enhancing proliferation, survival, cytolytic activity, and IFNγ production. The ability to induce potent antitumor activity by stimulating 4–1BB on tumor-specific cytotoxic T cells makes 4–1BB an attractive target for designing novel immuno-oncology therapeutics. To minimize systemic immune toxicities and enhance activity at the tumor site, we have developed a novel bispecific antibody that stimulates 4–1BB function when co-engaged with the tumor-associated antigen 5T4. ALG.APV-527 was built on the basis of the ADAPTIR bispecific platform with optimized binding domains to 4–1BB and 5T4 originating from the ALLIGATOR-GOLD human single-chain variable fragment library. The epitope of ALG.APV-527 was determined to be located at domain 1 and 2 on 4–1BB using X-ray crystallography. As shown in reporter and primary cell assays in vitro, ALG.APV-527 triggers dose-dependent 4–1BB activity mediated only by 5T4 crosslinking. In vivo, ALG.APV-527 demonstrates robust antitumor responses, by inhibiting growth of established tumors expressing human 5T4 followed by a long-lasting memory immune response. ALG.APV-527 has an antibody-like half-life in cynomolgus macaques and was well tolerated at 50.5 mg/kg. ALG.APV-527 is uniquely designed for 5T4-conditional 4–1BB–mediated antitumor activity with potential to minimize systemic immune activation and hepatotoxicity while providing efficacious tumor-specific responses in a range of 5T4-expressing tumor indications as shown by robust activity in preclinical in vitro and in vivo models. On the basis of the combined preclinical dataset, ALG.APV-527 has potential as a promising anticancer therapeutic for the treatment of 5T4-expressing tumors.

Supplementary figures and methods

Purpose: Local administration of immune-activating antibodies may increase the efficacy and reduce the immune-related adverse events associated with systemic immunotherapy of cancer. Here, we report the development and affinity maturation of a fully human agonistic CD40 antibody (IgG1), ADC-1013.Experimental Design: We have used molecular engineering to generate an agonistic antibody with high affinity for CD40. The functional activity of ADC-1013 was investigated in human and murine in vitro models. The in vivo effect was investigated in two separate bladder cancer models, both using human xenograft tumors in immune deficient NSG mice and using a syngeneic bladder cancer model in a novel human CD40 transgenic mouse.Results: Activation of dendritic cells (DC) by ADC-1013 results in upregulation of the costimulatory molecules CD80 and CD86, and secretion of IL12. ADC-1013 also activates DCs from human CD40 transgenic mice, and peptide-pulsed and ADC-1013–stimulated DCs induce antigen-specific T-cell proliferation in vitro. In vivo, treatment with ADC-1013 in a syngeneic bladder cancer model, negative for hCD40, induces significant antitumor effects and long-term tumor-specific immunity. Furthermore, ADC-1013 demonstrates significant antitumor effects in a human bladder cancer transplanted into immunodeficient NSG mice.Conclusions: Our data demonstrate that ADC-1013 induces long-lasting antitumor responses and immunologic memory mediated by CD40 stimulation. To the best of our knowledge, ADC-1013 represents the first immunomodulatory antibody developed for local immunotherapy of cancer. Clin Cancer Res; 21(5); 1115–26. ©2014 AACR.See related commentary by Dronca and Dong, p. 944

Supplementary Figure 3. Anti-tumor effect on MB49 tumors. ADC-1013 generates a significant anti-tumor effect on CD40 negative MB49 cells in hCD40tg mice. Treatment with ADC-1013 or isotype control was performed on day 7 and 10 post tumor inoculation. Increasing doses of ADC-1013 were administred peritumorally in 100 ul PBS and the individual tumor growth curves are displayed.

Supplementary Figure 2. Microphotographs from cryo sections showing representative images of A) Spleen, B) Liver and C) Brain (one slide representing data from one female and one male mice) from hCD40+ mice stained with an anti-human CD40 antibody or anti mouse CD40 antibody.

Supplementary Figure 1. A) Annexin V positive moDC after ADC-1013 stimulation and B) the effect of cross-linking on percentage of Annexin stained cells (n=6).

Supplementary Figure 6. Splenocytes from complete responders (CR) from ADC-1013 treatment of MB40 tumors were transplanted to naive mice. The receiving naïve mice were then challenged with MB49 tumors and the tumor volume followed over time until the first mice was sacrificed (**p

Supplementary Figure 5. hCD40tg mice with one tumor on each flank were treated peritumorally in the tumor on the right flank. Tumor volumes of individual untreated tumors (red) and treated tumors (black) are displayed.

Supplementary Methods

Supplementary Table 1. Selection strategy Supplementary Table 2. Kinetic parameters for ADC-1013 and B44 measured by Biacore Supplementary Table 3. In vitro potency (EC50) of ADC-1013 in DC and B cell assays Supplementary table 4. Phenotypic profiling of hCD40tg mice

Supplementary Figure 4. Anti-tumor effect on MB49 tumors. ADC-1013 generates a significant anti-tumor effect on CD40 negative MB49 cells in hCD40tg mice. Treatment with ADC-1013 or isotype control was performed on day 7 and 10 post tumor inoculation. Increasing doses of ADC-1013 were administred peritumorally in 100 ul PBS and the individual tumor growth curves are displayed.

4–1BB (CD137) is an activation-induced costimulatory receptor that regulates immune responses of activated CD8 T and natural killer cells, by enhancing proliferation, survival, cytolytic activity, and IFNγ production. The ability to induce potent antitumor activity by stimulating 4–1BB on tumor-specific cytotoxic T cells makes 4–1BB an attractive target for designing novel immuno-oncology therapeutics. To minimize systemic immune toxicities and enhance activity at the tumor site, we have developed a novel bispecific antibody that stimulates 4–1BB function when co-engaged with the tumor-associated antigen 5T4. ALG.APV-527 was built on the basis of the ADAPTIR bispecific platform with optimized binding domains to 4–1BB and 5T4 originating from the ALLIGATOR-GOLD human single-chain variable fragment library. The epitope of ALG.APV-527 was determined to be located at domain 1 and 2 on 4–1BB using X-ray crystallography. As shown in reporter and primary cell assays in vitro, ALG.APV-527 triggers dose-dependent 4–1BB activity mediated only by 5T4 crosslinking. In vivo, ALG.APV-527 demonstrates robust antitumor responses, by inhibiting growth of established tumors expressing human 5T4 followed by a long-lasting memory immune response. ALG.APV-527 has an antibody-like half-life in cynomolgus macaques and was well tolerated at 50.5 mg/kg. ALG.APV-527 is uniquely designed for 5T4-conditional 4–1BB–mediated antitumor activity with potential to minimize systemic immune activation and hepatotoxicity while providing efficacious tumor-specific responses in a range of 5T4-expressing tumor indications as shown by robust activity in preclinical in vitro and in vivo models. On the basis of the combined preclinical dataset, ALG.APV-527 has potential as a promising anticancer therapeutic for the treatment of 5T4-expressing tumors.

Background The CTLA-4 blocking antibody ipilimumab has demonstrated substantial and durable effects in patients with melanoma. While CTLA-4 therapy, both as monotherapy and in combination with PD-1 targeting therapies, has great potential in many indications, the toxicities of the current treatment regimens may limit their use. Thus, there is a medical need for new CTLA-4 targeting therapies with improved benefit-risk profile. Methods ATOR-1015 is a human CTLA-4 x OX40 targeting IgG1 bispecific antibody generated by linking an optimized version of the Ig-like V-type domain of human CD86, a natural CTLA-4 ligand, to an agonistic OX40 antibody. In vitro evaluation of T-cell activation and T regulatory cell (Treg) depletion was performed using purified cells from healthy human donors or cell lines. In vivo anti-tumor responses were studied using human OX40 transgenic (knock-in) mice with established syngeneic tumors. Tumors and spleens from treated mice were analyzed for CD8+ T cell and Treg frequencies, T-cell activation markers and tumor localization using flow cytometry. Results ATOR-1015 induces T-cell activation and Treg depletion in vitro. Treatment with ATOR-1015 reduces tumor growth and improves survival in several syngeneic tumor models, including bladder, colon and pancreas cancer models. It is further demonstrated that ATOR-1015 induces tumor-specific and long-term immunological memory and enhances the response to PD-1 inhibition. Moreover, ATOR-1015 localizes to the tumor area where it reduces the frequency of Tregs and increases the number and activation of CD8+ T cells. Conclusions By targeting CTLA-4 and OX40 simultaneously, ATOR-1015 is directed to the tumor area where it induces enhanced immune activation, and thus has the potential to be a next generation CTLA-4 targeting therapy with improved clinical efficacy and reduced toxicity. ATOR-1015 is also expected to act synergistically with anti-PD-1/PD-L1 therapy. The pre-clinical data support clinical development of ATOR-1015, and a first-in-human trial has started (NCT03782467). Electronic supplementary material The online version of this article (10.1186/s40425-019-0570-8) contains supplementary material, which is available to authorized users.

Purpose: Local administration of immune-activating antibodies may increase the efficacy and reduce the immune-related adverse events associated with systemic immunotherapy of cancer. Here, we report the development and affinity maturation of a fully human agonistic CD40 antibody (IgG1), ADC-1013. Experimental Design: We have used molecular engineering to generate an agonistic antibody with high affinity for CD40. The functional activity of ADC-1013 was investigated in human and murine in vitro models. The in vivo effect was investigated in two separate bladder cancer models, both using human xenograft tumors in immune deficient NSG mice and using a syngeneic bladder cancer model in a novel human CD40 transgenic mouse. Results: Activation of dendritic cells (DC) by ADC-1013 results in upregulation of the costimulatory molecules CD80 and CD86, and secretion of IL12. ADC-1013 also activates DCs from human CD40 transgenic mice, and peptide-pulsed and ADC-1013–stimulated DCs induce antigen-specific T-cell proliferation in vitro. In vivo, treatment with ADC-1013 in a syngeneic bladder cancer model, negative for hCD40, induces significant antitumor effects and long-term tumor-specific immunity. Furthermore, ADC-1013 demonstrates significant antitumor effects in a human bladder cancer transplanted into immunodeficient NSG mice. Conclusions: Our data demonstrate that ADC-1013 induces long-lasting antitumor responses and immunologic memory mediated by CD40 stimulation. To the best of our knowledge, ADC-1013 represents the first immunomodulatory antibody developed for local immunotherapy of cancer. Clin Cancer Res; 21(5); 1115–26. ©2014 AACR. See related commentary by Dronca and Dong, p. 944

The most important goals for the field of immuno-oncology are to improve the response rate and increase the number of tumor indications that respond to immunotherapy, without increasing adverse side effects. One approach to achieve these goals is to use tumor-directed immunotherapy, i.e., to focus the immune activation to the most relevant part of the immune system. This may improve anti-tumor efficacy as well as reduce immune-related adverse events. Tumor-directed immune activation can be achieved by local injections of immune modulators in the tumor area or by directing the immune modulator to the tumor using bispecific antibodies. In this review, we focus on therapies targeting checkpoint inhibitors and co-stimulatory receptors that can generate tumor-specific T cell responses through localized immune activation.

ATOR-1144 is a human CTLA-4 x GITR bispecific IgG1 antibody generated by fusing a high affinity CTLA-4 binder, derived by FIND® optimization of the CTLA-4 binding domain of CD86, to an agonistic GITR antibody isolated from the human antibody library ALLIGATOR-GOLD®. CTLA-4 and GITR are highly expressed on Tregs in the tumor microenvironment, and GITR is also expressed on NK cells and certain tumor cells. ATOR-1144 was designed to induce a tumor-directed immune activation for treatment of solid tumors and hematological malignancies. The in vitro activity of ATOR-1144 in terms of Treg depletion and activation of T cells and NK cells was investigated using purified cells from healthy human donors. ATOR-1144 induced depletion of primary Tregs in an ADCC assay with NK cells. T-cell activation in terms of IL-2 and IFN-γ release was demonstrated in the presence of CTLA-4 and Fcγ receptor crosslinking. NK cells treated with ATOR-1144 released IFN-γ, perforin and granzyme B, and displayed enhanced cytolytic killing of tumor cells. Moreover, NK cell-mediated depletion of CTLA-4-expressing cells was significantly improved upon stimulation with ATOR-1144 compared to monospecific anti-CTLA-4, indicating that NK cells activated via GITR acquire an enhanced capacity to induce ADCC. In conclusion, ATOR-1144 is a first-in-class bispecific tumor directed antibody targeting CTLA-4 and GITR. ATOR-1144 acts through several mechanisms, including depletion of Tregs, activation of effector T cells and activation of NK cells for enhanced Treg depletion and tumor cell killing. Citation Format: Sara Fritzell, Mattias Levin, Ida Åberg, Maria Johansson, Magnus Winnerstam, Karin Enell Smith, Peter Ellmark, Christina Furebring, Per Norlén, Anne Kvarnhammar. ATOR-1144 is a tumor-directed CTLA-4 x GITR bispecific antibody that acts by depleting Tregs and activating effector T cells and NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4077.

ATOR-1015 is a CTLA-4 x OX40 bispecific immune activating antibody developed for tumor-directed immunotherapy. ATOR-1015 binds both targets simultaneously, promoting cell-cell interactions expected to enhance the immuno-stimulating effect of the compound. The mode of action of ATOR-1015 is thought to be a combination of regulatory T cell (Treg) depletion and effector T cell activation. It can be seen as a next generation CTLA-4 antibody with tumor-directed activity and augmented Treg depletion. The ability to induce ADCC of human Treg was investigated using an FcγR expressing reporter assay demonstrating superior effect of ATOR-1015 compared to the monospecific antibody counterparts. Further, ATOR-1015 has been shown to induce activation of T cells in the presence of CTLA-4 expressing cells, ability that is not observed when combining the monoclonal counterparts. Syngeneic tumor models in vivo using human transgenic mice cross-reacting with both targets demonstrate that ATOR-1015 reduces tumor growth and prolongs survival. Further, ATOR-1015 treatment demonstrates superior increase in the intratumoral CD8+ T cell/Treg ratio compared to the monospecific counterparts, without affecting systemic T cells. This tumor directed immune activation is demonstrated to be due to the tumor localization abilities of ATOR-1015. In conclusion, ATOR-1015 is a next generation CTLA-4 antibody with tumor directed activity with augmented T-reg depletion. It is currently in GLP manufacturing of clinical material and will start clinical trials in the second half of 2018. Citation Format: Niina Veitonmäki, Mia Thagesson, Doreen Werchau, Karin Hägerbrand, Kristine Smedenfors, Anne Månsson-Kvarnhammar, Anna Rosén, Maria Johansson, Christina Furebring, Per Norlén, Peter Ellmark. The CTLA-4 x OX40 bispecific antibody ATOR-1015 induces anti-tumor effects through tumor-directed immune activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3623.

Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is a protein that binds and blocks the C5a receptor (C5aR) and formylated peptide receptor, thereby inhibiting the immune cell recruitment associated with inflammation. If CHIPS was less reactive with existing human antibodies, it would be a promising anti-inflammatory drug candidate. Therefore, we applied directed evolution and computational/rational design to the CHIPS gene in order to generate new CHIPS variants displaying lower interaction with human IgG, yet retaining biological function. The optimization was performed in four rounds: one round of random mutagenesis to add diversity into the CHIPS gene and three rounds of DNA recombination by Fragment INduced Diversity (FIND((R))). Every round was screened by phage selection and/or ELISA for decreased interaction with human IgG and retained C5aR binding. The mean binding of human anti-CHIPS IgG decreased with every round of evolution. For further optimization, new amino acid substitutions were introduced by rational design, based on the mutations identified during directed evolution. Finally, seven CHIPS variants with low interaction with human IgG and retained C5aR blocking capacity could be identified.

Stimulation of CD40 on dendritic cells to expand and activate tumor-specific T cells and generate anticancer immunity is an attractive therapeutic approach. Since CD40 agonists exert their effects upstream of checkpoint inhibitors, including PD-1 or PD-L1 antagonists, they are ideal candidates for combination regimens.

Procarboxypeptidase U [proCPU, thrombin-activatable fibrinolysis inhibitor (TAFI), EC 3.4.17.20] belongs to the metallocarboxypeptidase family and is a zymogen found in human plasma. ProCPU has been proposed to be a molecular link between coagulation and fibrinolysis. Upon activation of proCPU, the active enzyme (CPU) rapidly becomes inactive due to its intrinsic instability. The inherent instability of CPU is likely to be of major importance for the in vivo down-regulation of its activity, but the underlying structural mechanisms of this fast and spontaneous loss of activity of CPU have not yet been explained, and they severely inhibit the structural characterization of CPU. In this study, we screened for more thermostable versions of CPU to increase our understanding of the mechanism underlying the instability of CPU's activity. We have shown that single as well as a few 2-4 mutations in human CPU can prolong the half-life of CPU's activity at 37 degrees C from 0.2 h of wild-type CPU to 0.5-5.5 h for the mutants. We provide evidence that the gain in stable activity is accompanied by a gain in thermostability of the enzyme and increased resistance to proteolytic digest by trypsin. Using one of the stable mutants, we demonstrate the importance of CPU stability over proCPU concentration in down-regulating fibrinolysis.

The emerging field of proteomics has created a need for new high-throughput methodologies for the analysis of gene products. An attractive approach is to develop systems that allow for clonal selection of interacting protein pairs from large molecular libraries. In this study, we have characterized a novel approach for identification and selection of protein-protein interactions, denoted SPIRE (selection of protein interactions by receptor engagement), which is based on a mammalian expression system. We have demonstrated proof of concept by creating a general plasma membrane bound decoy receptor, by displaying a protein or a peptide genetically fused to a trunctated version of the CD40 molecule. When this decoy receptor is engaged by a ligand to the displayed protein/peptide, the receptor expressing cell is rescued from apoptosis. To design a high-throughput system with a highly parallel capacity, we utilized the B cell line WEHI-231, as carrier of the decoy receptor. One specific peptide-displaying cell could be identified and amplified, based on a specific receptor engagement, in a background of 12 500 wild-type cells after four selections. This demonstrates that the approach may serve as a tool in post-genomic research for identifying protein-protein interactions, without prior knowledge of either component.

Antibody evolution in vivo proceeds mainly by stepwise improvements, accomplished by single base pair substitutions. Lately, receptor revision, i.e. exchange of large parts of the V gene for another sequence, has been suggested to provide a complementary route for affinity maturation. By employing a receptor revision like evolution process in vitro using combinatorial libraries and phage display selection, we demonstrate here that maturation of a clone may preferentially proceed through exchange of a large gene segment rather than via minor sequence changes. These modifications of a CD40-specific human antibody fragment outline how receptor revision like events may provide an advantage to a particular clonotype put under selective pressure.

Immunoglobulin heavy chain (IgH) gene expression is guided by cis-regulatory elements which direct correct temporal and spatial expression in B lineage cells. One of these cis-acting elements is the IgH HS1,2 enhancer and previous studies in transgenic mice have revealed a temporally restricted activity of an HS1,2 enhancer-linked human β-globin reporter gene in B lineage cells. To assess whether this enhancer can impose strict temporal regulation onto a V H -promoter-Cμ reporter gene, transgenic mice were generated. These mice expressed high serum levels of protein from the transgene. Moreover, high levels of transgene expression were observed in spleen and thymus, while lower expression was found in heart and kidney and no expression was detected in liver and brain. Interestingly, transgene expression was confined to large, activated B cells and peritoneal B cells but not observed in small, resting splenic B cells or activated T cells. However, upon mitogenic stimulation of resting B cells with LPS, high levels of transgene expression was induced. Our data demonstrate that the HS1,2 enhancer can interact with a natural V H promoter in a strict temporal fashion and when provided with an appropriate activation signal, this V H promoter/enhancer construct can induce transgene expression in resting B, but not T lineage cells. Our data are compatible with a model whereby the regulation of IgH gene expression may be subject to regulation by distinct subsets of cis-regulatory elements acting at different stages of B lymphocyte development. Thus, Ig gene expression may be regulated via an interaction between the V H promoter and 3′ enhancer elements (here typified by the HS1,2 enhancer) in terminally differentiated B lineage cells.

The Chemotaxis Inhibitory Protein of Staphylococcus aureus (CHIPS) binds and blocks the C5a receptor (C5aR) and formyl-peptide receptor (FPR). This way, CHIPS is a potent inhibitor of the immune cell recruitment associated with inflammation. Truncation of the protein and the introduction of mutations, shifts the expression towards the insoluble fraction of Escherichia coli, whereas the wild-type protein can be solubly expressed. A protocol for expression and tag independent purification of biologically active CHIPS variants has been established to enable further characterization of an improved CHIPS variant, called ADC-1004. The CHIPS variants were purified by washing of E. coli inclusion bodies followed by refolding and gel filtration. New techniques were utilized to optimize the purification process. Expression in inclusion bodies was increased by the use of Ultra Yield™ flasks and optimal refolding conditions were determined by the use of the iFOLD Refolding System 2™. The folding and biological activity of the purified proteins were analyzed by circular dichroism (CD) spectroscopy and flow cytometry, respectively, and compared to solubly produced CHIPS31–113 and wild-type CHIPS1–121. We show that the CHIPS variants produced in inclusion bodies can be refolded and purified to achieve equal biological activity as solubly produced CHIPS31–113 and wild-type CHIPS1–121. The truncation causes minor structural changes while purification from inclusion bodies or the soluble fraction does not further affect the structure. (Less)

Human antibodies directed towards functionally associated tumor antigens have great potentials as adjuvant treatment in cancer therapy. Here we describe an efficient subtractive approach to select single chain Fv (scFv) antibodies, specifically binding to unknown rapidly internalizing tumor-associated antigens (TAA) on human breast and pancreatic carcinoma cell lines. After re-engineering the scFv into intact IgG molecules, these fully human antibodies displayed individual binding profiles to TAAs on breast, pancreatic, colorectal and prostate carcinomas, while showing no reactivity to lymphomas. The ability of the selected antibodies to undergo receptor-mediated internalization was verified by confocal microscopy, thus proving our strategy to provide a unique set of human antibodies, potentially useful in immunotherapy.

CD40 is a tumour necrosis factor receptor (TNFR) family member of central importance for the adaptive immune system. To elucidate the functional role of the different extracellular domains of CD40, we have created a set of truncated CD40 molecules where domains, or parts of domains, have been removed. These CD40 proteins, which contain a peptide tag in the N-terminal end, have been expressed in a murine B-cell line, WEHI 231. It was found that ligation of these engineered CD40 proteins via the peptide tag, was sufficient to rescue as well as to promote proliferation of apoptotic WEHI 231 cells, even when all the extracellular domains of CD40 were absent. Our results suggest that pre association of CD40 in the cell membrane plays no critical role for the CD40 signalling pathway. Furthermore, our data imply that conformational changes initiated in the extracellular domains of CD40 are not essential for signal transduction.

The CD40-CD40L interaction plays a critical role in both humoral and cellular immune responses and interfering antibodies have been suggested as an effective approach for the treatment of lymphomas and autoimmune diseases. In this study we have profiled a panel of mouse antihuman CD40 monoclonal antibodies (MoAbs), regarding their CD40 binding affinity and epitope-specificity relative to the CD40L binding in relation to their cellular activating potential. Despite a rather similar domain-recognition profile, the MoAbs blocked the CD40L binding to a varying degree, with MoAb 5C3 being the poorest inhibitor. There was no correlation between affinity and cellular activation potential. In contrast, a correlation between the ability to block CD40L-binding and activation potential could be seen. We believe that this analysis of several mouse anti-CD40 antibodies can be used to develop strategies for producing new human anti-CD40 antibodies that can more effectively induce or block B-cell proliferation.

CD40 plays a central regulatory role in the immune system and antibodies able to modulate CD40 signalling may consequently have a potential in immunotherapy, in particular for treatment of lymphomas and autoimmune disease like multiple sclerosis. As a first step to achieve this goal, we describe the selection and characterization of a novel set of fully human anti-CD40 antibody fragments (scFv) from a phage display library (n-CoDeR). In order to determine their biological potential, these antibody fragments have been analysed for their ability to promote B-cell activation, rescue from apoptosis and to block the CD40-CD40 ligand (CD40L) interaction. The selected cohort of human scFv could be subcategorized, each expressing a distinct functional signature. Thus scFv were generated that induced B-cell proliferation, rescued B cells from apoptosis and blocked the CD40-CD40L interaction to different extents. In particular, one of the scFv clones (F33) had the ability to abrogate completely this interaction. The epitope recognition patterns as well as individual rate constants were also determined and the affinity was shown to vary from low to high nanomolar range. In conclusion, this panel of human anti-CD40 scFv fragments displays a number of distinct properties, which may constitute a valuable source when evaluating candidates for in vivo trials.

Antibodies can neutralize the infectious properties of human cytomegalovirus (CMV). In vivo, the major neutralization determinants are located on glycoprotein B (gB). Recombinant human antibodies, that carry different constant regions (IgG1, IgG3 and the synthetic variant IgG3mA) against two of these epitopes were investigated for their ability to recruit the complement cascade for destruction of the virus. It was shown that all variants of an antibody against the antigenic domain (AD)-2 epitope displayed a similar neutralization activity despite the fact that improved C1q binding was observed for IgG3 and IgG3mA over the IgG1 variant. In contrast, an antibody against the AD-1 epitope carrying the normal IgG3 constant region, was less efficient than its IgG1 counterpart in neutralizing the virus in the absence of complement. However, it restored its activity in the presence of complement to the level of the naturally occurring IgG1 version. The same antibody was substantially more potent in neutralizing the virus in the presence of complement if it carried the IgG3mA constant region. This demonstrates the importance of the constant domain for the biological activity of AD-1 specific antibodies, a factor that should be taken into account when using antibody-based therapeutics or when inducing antibodies by vaccination.

Background: The wide range of antibody specificity and affinity results from the differing shapes and chemical compositions of their binding sites. These shapes range from discrete grooves in antibodies elicited by linear oligomers of nucleotides and carbohydrates to shallow depressions or flat surfaces for accommodation of proteins: peptides and large organic compounds. Objectives: To determine the Fab structure of a high-affinity human antitoxin antibody. To explore structural features which enable the antibody to bind to intact tetanus toxoid, peptides derived from the sequence of the natural immunogen and antigenic mimics identified by combinatorial chemistry. To explain why this Fab shows a remarkable tendency to produce crystals consistently diffracting to d spacings of 1.7-1.8 A. To use this information to engineer a strong tendency to crystallize into the design of other Fabs. Study design: The protein was crystallized in hanging or sitting drops by a microseeding technique in polyethylene glycol (PEG) 8000. Crystals were subjected to X-ray analysis and the three-dimensional structure of the Fab was determined by the molecular replacement method. Interactive computer graphics were employed to fit models to electron density maps, survey the structure in multiple views and discover the crystal packing motif of the protein. Results: Exceptionally large single crystals of this protein have been obtained, one measuring 5 x 3 x 2 mm (l x w x d). The latter was cut into six irregular pieces, each retaining the features of the original in diffracting to high resolution (1.8 A) with little decay in the X-ray beam. In an individual Fab, the active site is relatively flat and it seems likely that the protein antigen and derivative peptides are tightly held on the outer surface without significant penetration into the interior. There is no free space to accommodate even a dipeptide between V(H) and V(L). One of the unique features of the B7-15A2 Fab is a large aliphatic ridge dominating the center of the active site. The CDR3 of the H chain contributes significantly to this ridge, as well as to adjoining regions projected to be important for the docking of the antigen. Both the ease of crystallization and the favorable diffraction properties are mainly attributable to the tight packing of the protein molecules in the crystal lattice. Discussion: The B7-15A2 active site provides a stable and well defined platform for high affinity docking of proteins, peptides and their mimotopes. The advantages for future developments are suggested by the analysis of the crystal properties. It should be possible to incorporate the features promoting crystallization, close packing and resistance to radiation damage into engineered human antibodies without altering the desired specificities and affinities of their active sites.

A novel mammalian eukaryotic expression vector for the production of immunoglobulin heavy chain (IgH) genes has been designed. This expression vector contains the variable heavy chain (VH) promoter, the IgH intron enhancer (μE) and the IgH 3′ enhancer (3′E). This construct, designated pTIF-1, was stably transfected into the myeloma cell line J558L. A fivefold increase in the expression level of a rearranged IgH gene was observed when using the pTIF-1 vector containing the 3′E compared to an expression vector lacking this enhancer. Interestingly, this positive effect on the expression level of the 3′ enhancer appears to be position independent. The introduction of two recently identified Ig control elements, HS3 and HS4, to the vector cassette did not further elevate the expression level in the cell line tested. The pTIF-1 vector can be used for expression of any antibody specificity, using PCR amplification of the VDJ region of interest. Furthermore, the constant region can easily be exchanged, which further facilitates studies to dissect different effector functions of IgH constant genes.

Background The Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) blocks the Complement fragment C5a receptor (C5aR) and formylated peptide receptor (FPR) and is thereby a potent inhibitor of neutrophil chemotaxis and activation of inflammatory responses. The majority of the healthy human population has antibodies against CHIPS that have been shown to interfere with its function in vitro. The aim of this study was to define potential epitopes for human antibodies on the CHIPS surface. We also initiate the process to identify a mutated CHIPS molecule that is not efficiently recognized by preformed anti-CHIPS antibodies and retains anti-inflammatory activity. Results In this paper, we panned peptide displaying phage libraries against a pool of CHIPS specific affinity-purified polyclonal human IgG. The selected peptides could be divided into two groups of sequences. The first group was the most dominant with 36 of the 48 sequenced clones represented. Binding to human affinity-purified IgG was verified by ELISA for a selection of peptide sequences in phage format. For further analysis, one peptide was chemically synthesized and antibodies affinity-purified on this peptide were found to bind the CHIPS molecule as studied by ELISA and Surface Plasmon Resonance. Furthermore, seven potential conformational epitopes responsible for antibody recognition were identified by mapping phage selected peptide sequences on the CHIPS surface as defined in the NMR structure of the recombinant CHIPS31–121 protein. Mapped epitopes were verified by in vitro mutational analysis of the CHIPS molecule. Single mutations introduced in the proposed antibody epitopes were shown to decrease antibody binding to CHIPS. The biological function in terms of C5aR signaling was studied by flow cytometry. A few mutations were shown to affect this biological function as well as the antibody binding. Conclusion Conformational epitopes recognized by human antibodies have been mapped on the CHIPS surface and amino acid residues involved in both antibody and C5aR interaction could be defined. This information has implications for the development of an effective anti-inflammatory agent based on a functional CHIPS molecule with low interaction with human IgG.

Christina Furebring a,1, Andrea Speckner b,1, Michael Mach b, Inger Sandlie c, Lars Norderhaug c, Carl A.K. Borrebaeck a, Helene Turesson a, Mats Ohlin a,∗ a Department of Immunotechnology, Lund University, Lund, Sweden b Institut fur Klinische und Molekulare Virologie, Friedrich-Alexander Universitat Erlangen-Nurnberg, Erlangen, Germany c Division of Molecular and Cell Biology, Department of Biology, University of Oslo, Oslo, Norway

The relation between domain structures of recombinant monoclonal antibody fragments and their reaction kinetics was studied for the first time using a novel biosensor based on surface plasmon resonance technology. The association and dissociation rate constants of Fab, Fv and single domain (VH fragment) anti-lysozyme antibodies were determined and compared to the intact monoclonal antibody. Fab and Fv fragments showed similar reaction kinetics and had affinity constants of 6 x 10(9) M-1 and 25 x 10(9) M-1, respectively. The single domain antibody had significantly different reaction kinetics compared to the fragments consisting of paired heavy and light chain domains. The VH domain had both a higher dissociation and a lower association rate constant, which resulted in an affinity constant approximately 250 times lower than the Fab fragment. This rapid evaluation of antibody reaction kinetics should prove to be an important selection parameter when comparing antibody fragments for their utility in therapeutic or other applications.

To delineate the role of the IgH 3' enhancer in the regulation of Ig heavy (IgH) chain gene expression, mice harbouring rearranged IgH transgenes, with (PSVmu3) and without (PSVmu1) this element, were produced. RNA and protein analysis from the different transgenic lines revealed a 5- to 7-fold increase in the expression level of the transgene containing the IgH 3' enhancer. This difference is also reflected at the protein level in hybridomas generated from the two transgenic lines. The elevation of transgene Ig expression in the PSVmu3 lines is restricted to activated B lymphocytes, an observation which is further supported by the ability of this transgene to be reactivated upon immunization. Interestingly, although the up-regulation of transgene expression in PSVmu3 animals is considerably higher in comparison to the PSVmu1 animals, a significant response is still observed in the PSVmu1 mice. We speculate therefore that the IgH locus is subject to transcriptional modification in late B cell development. Our data suggest that both the Emu enhancer and the IgH 3' enhancer can up-regulate transgene Ig expression, but the presence of the 3' enhancer results in elevated levels of transgene Ig production. It therefore appears that the expression level of IgH genes is subject to transcriptional modification during B cell development. Additional control elements are most likely required for optimal Ig expression, since our expression data from the transgene in PSVmu3 animals are incompatible with endogenous Ig levels. The recent identification of additional enhancer elements in the far 3' end of the IgH locus supports this possibility. The data presented here provides a sound basis for the production of high levels of mAb, possibly tailored to suit the needs of the researcher.

Local administration of immune activating antibodies may increase the efficacy and reduce the immune-related adverse events associated with systemic immunotherapy of cancer. Here we report the development of a fully human agonistic CD40 antibody (IgG1), ADC-1013, which has been optimized for local immunotherapy by increasing potency and tumor retention. ADC-1013 activates CD40 receptors on antigen-presenting cells such as dendritic cells, resulting in up-regulation of the co-stimulatory molecules CD80 and CD86, and induction of IL-12. In addition, ADC-1013 induces direct tumor killing of CD40+ tumors, e.g. via antibody-dependent cellular cytotoxicity (ADCC). The anti-tumor effects of ADC-1013 were first assessed in a bladder cancer model (EJ) in immunodeficient NSG mice. Significant anti-tumor responses were demonstrated, and further augmented in mice repopulated with human moDCs/T cells. To study the anti-tumor effects related to the immune activating properties of ADC-1013 in more detail, a human CD40 positive transgenic mouse (hCD40tg) in C57/BL-6 background was used. This transgenic mouse strain has an intact immune system and fully functional dendritic cells that are activated upon ADC-1013 treatment. Furthermore, the dendritic cells obtained from this strain are able to induce antigen specific T cell activation in vitro upon stimulation with ADC-1013. Importantly, treatment with ADC-1013 in a syngeneic bladder cancer (MB49) model, which is hCD40 negative, demonstrated that ADC-1013 induce significant tumor protection and long term immunity independent of direct tumor targeting. In addition, the anti-tumor immunity was shown to be T-cell dependent. To our knowledge, ADC-1013 represents the first immunomodulatory antibody optimized for local immunotherapy of cancer. It is currently in late pre-clinical development and will enter clinical trials in 2014.

Local activation of costimulatory pathways by e.g. CD40 activation has been shown to generate powerful systemic anti-tumor responses. Here we report significant anti-tumor effects obtained with an optimized fully human agonistic CD40 antibody, ADC-1013, in two syngeneic tumor models. An hCD40 transgenic mouse (hCD40tg) strain was used to evaluate the immune mediated anti-tumor effects of ADC-1013. Dendritic cells obtained from hCD40tg mice were hCD40 positive and could be activated by stimulation with ADC-1013 to a similar extent as human monocyte derived dendritic cells. Furthermore, stimulation of dendritic cells from hCD40tg mice in vitro, with ADC-1013, induced antigen specific T cell activation. Two different syngeneic tumor models, hCD40 negative MB49 bladder cancer and hCD40 positive B16.F10.hCD40+ melanoma, was used to demonstrate anti-tumor effects. Subcutaneous tumors from both models were characterized by flow cytometry and immunohistochemistry in hCD40tg mice. Treatment of the bladder cancer model (MB49) with ADC-1013 resulted in significant anti-tumor response and long term tumor immunity. The anti-tumor immunity was shown to be T cell dependent and naive mice were protected from tumor challenge by transplantation of splenocytes from cured hCD40tg mice. In addition, significant anti-tumor effect was demonstrated in a subcutaneous B16.F10.hCD40+ melanoma model. The human CD40 agonistic antibody ADC-1013 is the first costimulatory antibody optimized for local immunotherapy of cancer. Strong immune mediated anti-tumor effects were demonstrated. ADC-1013 is currently in late pre-clinical development.

Activation of CD40 on dendritic cells increases cross-presentation of tumor antigens and as a result the number of activated tumor directed T effector cells. CD40 agonists have been shown to generate powerful systemic anti-tumor responses when administered in situ. The pre-clinical proof of concept data has been generated mainly using an hCD40 transgenic mouse (hCD40tg) strain. Anti-tumor effects has been demonstrated in multiple syngeneic tumor models including bladder tumor (MB49), melanoma (B16) and lymphoma (A20) models. The anti-tumor effects has been shown to induce long term, T cell dependent, tumor immunity. As expected, the anti-tumor effects depends on the immune status of the tumors as evaluated by immunohistochemistry and flow cytometry. Since CD40 agonistic antibodies mainly exert their effects upstream of the checkpoint inhibitors they have the potential to be ideal candidates for combination regimens including e.g. PD-1 antagonists and OX40 antibodies. Combining ADC-1013 with antibodies against these targets has been shown to improve the anti-tumor effects in B16.F10 melanoma. Toxicology studies were performed in cynomolgus monkeys using subcutaneous or intravenous administration. Doses of 1 to 10 mg/kg were evaluated in both studies. ADC-1013 was well tolerated at all dose levels and no major safety concerns were identified. Receptor saturation of approximately 95% was achieved and pharmacodynamic activity, notably B-cell depletion, was observed at all dose levels. In cynomolgus monkey, serum concentrations of ADC-1013 declined in a multi-phasic manner following a single intravenous dose. The mean terminal half-life was approximately 30 hours. Following subcutaneous administration the mean elimination half-life was found to be between 0.6 and 3.2 days. The clearance (CL_F) decreased and the half-life increased with increasing dose indicating target-mediated clearance. Alligator Bioscience has recently started dosing of the first patients in a clinical trial of the CD40 agonistic antibody ADC-1013. The study is a first-in-human, multicenter, open-label, multiple ascending dose Phase I study in patients with advanced solid tumors to determine the safety, pharmacokinetics and pharmacodynamics of intratumorally administered ADC-1013.