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Binding to human colon cancer cells HCT 116 was analyzed by flow cytometry for purified antibody variants of lgGl-hDR5-01-G56T and lgGl-hDR5-05 with and without the E430G mutation. Single cell suspensions were prepared and binding was analyzed for serial dilution antibody preparation series (range 0.0006 to 10 μg/mL final concentrations in 4- fold dilutions) as described in Example 3. After incubation with the secondary antibody, cells were washed twice, resuspended in 100 μί FACS buffer, and antibody binding was analyzed on a BD LRSFFortessa cell analyzer (BD Biosciences).

Bispecific IgGl antibodies were generated by Fab-arm-exchange under controlled reducing conditions. The basis for this method is the use https://cryptolisting.org/coin/wings/ of complementary CH3 domains, which promote the formation of heterodimers under specific assay conditions as described in WO2011/131746.

The combination of lgGl-DR5-01-K409R-E430G and lgGl-DR5-05-F405L- E430G was more potent than either antibody alone and more potent than the combination of the antibodies without the E430G mutation. The combination of lgGl-DR5-01-K409R and lgGl-DR5-05-F405L was more potent than either antibody alone. These data show that introduction of the hexamerization-enhancing mutation E430G resulted in enhanced induction of cell killing upon binding of the chimeric DR5 antibodies 01 and 05, both as single antibodies and in combination, with the combination being the most potent. Figure 4 shows that the antibodies lgGl-DR5-01-K409R-E430G and lgGl-DR5-05-F405L- E430G showed dose-dependent binding to Rhesus macaque DR5 expressed on CHO cells. Binding to CHO cells transfected with human DR5 and mock-transfected CHO cell was tested as positive and negative control, respectively.

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In one embodiment of the present invention the anti-DR5 antibody induces caspase-dependent cell death. Caspase-dependent cell death may be induced by activation of caspase-3 and/or caspase-7.

Examples of host cells include yeast, bacterial and mammalian cells, such as CHO or HEK-293 cells. For example, the host cell may comprise a nucleic acid stably integrated into the cellular genome that comprises a sequence coding for expression of an antibody described herein. The host cell may comprise a nucleic acid stably integrated into the cellular genome that comprise a sequence coding for expression of a first or a second polypeptide described herein. Alternatively, the host cell may comprise a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, which comprises a sequence coding for expression of an antibody described herein.

Anti-DR5 antibody at a fixed concentration of e.g. 1 μg/mL may be added to adhered cells and incubated for 1 to 24 hours. Caspase-3/7 activation can be determined by using special kits for this purpose, such as the PE Active Caspase-3 Apoptosis Kit of BD Pharmingen (Cat nr ) (example 19 and 25) or the Caspase-Glo 3/7 assay of Promega (Cat nr G8091) (examples 20 and 45). Phosphatidylserine exposure and cell death can be determined by using special kits for this purpose, such as the FITC Annexin V Apoptosis Detection Kit I from BD Pharmingen (Cat nr ) (examples 19 and 25). In one embodiment the antibody is an anti-DR5 antibody and said anti-DR5 antibody is agonistic. That the antibody is agonistic is to be understood as that the antibody clusters, stimulates or activates DR5.

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Non-reduced CE-SDS data showed that the samples in the presence of arginine displayed high %HMW impurities that were not present in samples containing NaCI. Overall, the summation of the available data supports the choice of a formulation containing histidine and sodium chloride for this antibody. Caspase-3/7 activation was measured in time using the Caspase-Glo 3/7 assay, essentially as described in Example 20. Briefly, cells were harvested by trypsinization, passed through a cell strainer, pelleted by centrifugation for 5 minutes at 1,200 rpm and resuspended in culture medium at a concentration of 1.6x10s cells/mL.

Membrane proteins were expressed in Freestyle CHO-S cells (Life technologies), using the freestyle Max reagent, as described by the manufacturer. In one embodiment, the invention relates to a pharmaceutical composition according to the beat cop igg invention comprising one or more anti-DR5 antibodies for use in treatment of infectious disease, autoimmune disease or cardiovascular anomalies. Antibodies may be produced by use of recombinant eukaryotic or prokaryotic host cells.

25 μί of the single cell suspensions (4,000 cells per well) were seeded in 384-wells culture plates (Perkin Elmer, Cat nr ) and incubated overnight at 375C. 25 μί sample was added (26.6 nM final concentrations) and incubated for 1, 2, 4 and 6 hours at 375C.

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In one embodiment, an agonistic anti-DR5 antibody of the present invention bound to DR5 activates the same intracellular pathways as TRAIL bound to DR5. The epitope or binding region on the extracelluar domanin on human DR5 of the antibodies of the present invention may be determined by use of the method of domain-swaped DR5 moleues as described in Example 6. In brief, domain-swaped DR5 molecules are transiently expressed in CHO cells, binding of antibodies to the domain-swaped human DR5 molecules are determined by a FACS assay.

Briefly, 100 μί single cell suspensions (5,000 cells per well) were seeded in 96-well plates and at the same time, 50 μί of serial dilution antibody preparation series (range 0.0003 to 20 μg/mL final concentrations in 4-fold dilutions) were added and incubated for 3 days at 375C. Also the efficacy of the combination of lgGl-hDR5-01-G56T + lgGl-hDR5-05 was improved by introduction of the S440Y mutation in both antibodies, represented by the decreased EC50 (Figure 37B).

For the DoE stability study, the initial DSC results confirmed that 30mM histidine pH 6.0 formulations had higher TonSet melting temperature values. SEC data demonstrated that histidine pH 6.0 in the presence of charged excipients conferred the most stability for lgGl-hDR5-05-E430G. Results from icIEF showed that pH 5.5 and 6.0 samples how to buy procurrency were more resistant to changes in charge heterogeneity. DLS data showed that histidine pH 6.0 formulations in the presence of charged excipients had the least change in polydispersity. The results for the reduced CE-SDS showed that histidine pH 6.0 formulations in the presence of charged excipients were the best formulations.

• With all these models, several possible new drugs and therapy strategies were tested in the last years.
• Helper T cells oversee cytokine signaling to activate B cells and increase the efficiency of other immune cells, such as macrophages.
• Although the majority of patients with glaucoma have elevated intraocular pressure as the presumed etiology for their resultant neuropathy, it is well known that approximately 25% of patients with glaucoma have intraocular pressure within the normal range for their race.
• Human- mouse chimeric antibodies lgGl-DR5-01-K409R-E430G and lgGl-DR5-05-F405L-E430G were tested in flowcytometric analysis on FACS for binding to (A) mock-transfected CHO cells, (B) human DR5-transfected CHO cells and (C) Rhesus macaque DR5-transfected CHO cells.
• Dendritic cells can process and present most types of pathogens, such as viruses, bacteria, fungi and parasites.
• Dendritic cells are produced in bone marrow and migrate through the blood to tissues where they monitor for pathogens.

The F405L and K409R (EU numbering) mutations were introduced in anti- DR5 IgGl antibodies to create antibody pairs with complementary CH3 domains. The F405L mutation was introduced in lgGl-DR5-05 and lgGl-DR5-05-E430G; the K409R mutation was introduced in lgGl-DR5-01 and lgGl-DR5-01-E430G. To generate bispecific antibodies, the two parental complementary antibodies, each antibody at a final concentration of 0.5 mg/mL, were incubated with 75 mM 2-mercaptoethylamine-HCI (2-MEA) in a total volume of 100 μί TE at 31°C for 5 hours. The reduction reaction was stopped by removing the reducing agent 2-MEA using spin columns (Microcon centrifugal filters, 30k, Millipore) according to the manufacturer’s protocol. In this way the bispecific antibodies lgGl-DR5-01- K409R x lgGl-DR5-05-F405L (BsAb DR5-01-K409R x DR5-05-F405L) and lgGl-DR5-01-K409R- E430G x lgGl-DR5-05-F405L-E430G (BsAb DR5-01-K409R-E430G x DR5-05-F405L-E430G) were generated.

Antibodies to be formulated in a pharmaceutical composition of the invention may be produced recombinantly in a host cell by introducing an expression vector carrying sequences coding for the antibody chains. An expression vector in the context of the present invention may be any suitable vector, including chromosomal, non-chromosomal, and synthetic nucleic acid vectors (a nucleic acid sequence comprising a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid ( NA or DNA) vectors. In one embodiment of the present invention, the antibody in a pharmaceutical composition of the invention is an anti-DR5 antibody which engages into oligomerization such as hexamerization of antibodies on target cells expressing DR5. Oligomerization such as hexamerization is mediated through Fc-Fc interactions.

Viability was assessed as described in Example 11 with 1,000 cells per well and antibody concentrations series ranging from 0.0001 to 10,000 ng/mL final concentrations in 4-fold dilutions in a total volume of 200 μί. Figure 12C shows that introduction of the G56T mutation in lgGl-hDR5-01-E430G had no effect on the killing efficacy of the antibody in combination with lgGl-hDR5-05-E430G. Figure 7 shows that introduction of the E430G mutation enhanced the potency of the chimeric antibodies lgGl-DR5-01-K409R and lgGl-DR5-05-F405L in both COLO 205 (A) and HCT 116 (B) cells.

In one embodiment of the invention the anti-DR5 antibody induces caspase-3 and/or caspase-7 dependent cell death. In one embodiment of the present invention the antibody induces apoptosis.

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Initial baseline biophysical screening results suggested that acetate and histidine formulations at pH 5.5 were optimal buffer/pH conditions. Additionally, arginine and NaCI were better choice of excipients when compared to sorbitol and sucrose.

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After mixing by shaking for one minute at 500 rpm, the plates were incubated for one hour at room temperature. The effect of the hexamerization-enhancing mutation S440Y on the capacity of the single antibodies and the combination of lgGl-hDR5-01-G56T and lgGl-hDR5-05 to kill COLO 205 human colon cancer cells was studied in a viability assay. Cells were harvested and a CellTiter-Glo luminescent cell viability assay was performed as described in Example 8.

In example 19 it was described that incubation with the combination of chimeric DR5 antibodies lgGl-DR5-01-K409R-E430G + lgGl-DR5-05-F405L-E430G induced caspase-3 activation in COLO 205 colon cancer cells. The percentage of active caspase-3-positive cells was higher after 5 hours than after 24 hours of incubation with the antibody combination. In this example, Caspase-3/7 activation was measured in time using the Caspase-Glo 3/7 assay (Promega, Cat nr G8091), in which a substrate with the Caspase-3/7 recognition motif DEVD releases aminoluciferin, a substrate of luciferase, upon cleavage. Cells were harvested by pooling the culture supernatant containing non-adherent cells and trypsinized adherent COLO 205.

The in vivo anti-tumor efficacy of different doses lgGl-DR5-01-K409R-E430G + lgGl-DR5- 05-F405L-E430G was evaluated and compared to an equivalent dosing of IgGl-CONA in the subcutaneous COLO 205 human colon cancer xenograft model. Tumor cell inoculation, mice handling, tumor outgrowth measurements and endpoint determination were performed as described in Example 26. At day 10, the average tumor volume was ~400 mm3 and the mice were sorted into groups with equal tumor size variance (Table 3 below). Mice were treated by intravenous (i.v.) injection of 40 μg (2 mg/kg), 10 μg (0.5 mg/kg) or 2 μg (0.1 mg/kg) antibody in 100 μί PBS on day 10.

Plates were removed from the incubator to let the temperature decrease till room temperature. 25 μί supernatant was removed and replaced by 25 μί Caspase-Glo 3/7 Substrate.

Cells were passed through a cell strainer, pelleted by centrifugation for 5 minutes at 1,200 rpm and resuspended in culture medium at a concentration of 0.8x10s cells/mL. 25 μί of the single cell suspensions (2,000 cells per well) were seeded in 384-wells culture plates (Perkin Elmer, Cat nr ) and incubated for 16 hours at 375C. 25 μί antibody sample was added (1 μg antibody https://cryptolisting.org/ final concentration) and incubated for 1, 2, 5 and 24 hours at 375C. A viability assay was performed to compare the capacity of the combination of humanized antibodies lgGl-hDR5-01-G56T-E430G + lgGl-hDR5-05-E430G with the capacity of the combination of humanized antibodies lgGl-hDR5-01-E430G + lgGl-hDR5-05-E430G to induce killing of BxPC-3 pancreatic cancer cells.

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One method for determining this is by inhibiting Fc-Fc interactions which indicate that antibodies oligomerizies e.g. hexamerizies. The Fc-Fc interactions can be inhibited by a peptide binding to the hydrophobic patch beat cop igg involved in Fc-Fc interactions such as DCAWHLGELVWCT as described in example 15. In one embodiment the antibody is an anti-DR5 antibody and said anti-DR5 antibody induces programmed cell death in a target cell.